Oligoribonucleotide inhibitors of nrf2 and methods of use thereof for treatment of cancer

ABSTRACT

The invention provides novel double stranded oligoribonucleotides that inhibit the Nrf2 gene. The invention also provides a pharmaceutical composition comprising one or more such oligoribonucleotides, and a vector capable of expressing the oligoribonucleotide. The present invention also relates to methods and compositions for treating or preventing the incidence or severity of a cancerous disease, particularly various lung cancers.

This application claims benefit of U.S. Provisional Application No. 60/937,135, filed Jun. 26, 2007 and of U.S. Provisional Application No. 60/919,413, filed Mar. 21, 2007, which contents of both are hereby incorporated by reference into this application.

Throughout this application various patent and scientific publications are cited. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

This application contains nucleotide and/or amino acid sequences which are present on a compact disc labeled “OLIGORIBONUCLEOTIDE INHIBITORS OF NRF2 AND METHODS OF USE THEREOF FOR TREATMENT OF CANCER”. The file contained on this disc is named “Sequence Listing.txt”, is 347 KB in size, and was created on Mar. 19, 2008 in the IBM-PCT machine format and has operating system compatibility with MS-Windows. The entire content of this compact disc is hereby incorporated by reference into this application.

BACKGROUND OF THE INVENTION siRNAs and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, attempts to study this phenomenon and to manipulate mammalian cells experimentally were frustrated by an active, non-specific antiviral defense mechanism which was activated in response to long dsRNA molecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defense mechanisms (see Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747). As a result, small interfering RNAs (siRNAs), which are short double-stranded RNAs, have become powerful tools in attempting to understand gene function.

Thus, RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) or microRNAs (miRNAs) (Ambros V. Nature 431:7006, 350-355 (2004); and Bartel D P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs: genomics, biogenesis, mechanism, and function). The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. An siRNA is a double-stranded RNA molecule which down-regulates or silences (prevents) the expression of a gene/mRNA of its endogenous (cellular) counterpart. RNA interference is based on the ability of dsRNA species to enter a specific protein complex, where it is then targeted to the complementary cellular RNA and specifically degrades it. Thus, the RNA interference response features an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA may take place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al 2001, Genes Dev., 15, 188). In more detail, longer dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein et al., Nature, 2001, v. 409, p. 363-6; Lee et al., Nature, 2003, 425, p. 415-9). The RISC protein complex recognizes these fragments and complementary mRNA. The whole process is culminated by endonuclease cleavage of target mRNA (McManus&Sharp, Nature Rev Genet, 2002, v. 3, p. 737-47; Paddison &Hannon, Curr Opin Mol. Ther. 2003 June; 5(3): 217-24). For information on these terms and proposed mechanisms, see Bernstein E., Denli A M. Hannon G J: 2001 The rest is silence. RNA. I; 7(11): 1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 116; 107(4): 415-8 and PCT publication WO 01/36646 (Glover et al).

The selection and synthesis of siRNA corresponding to known genes has been widely reported; see for example Chalk A M, Wahlestedt C, Sonnhammner E L. 2004 Improved and automated prediction of effective siRNA Biochem. Biophys. Res. Commun. Jun 18; 319(1): 264-74; Sioud M, Leirdal M., 2004, Potential design rules and enzymatic synthesis of siRNAs, Methods Mol. Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004, Gene specific siRNA selector Bioinformatics. 112; 20(3): 430-2. and Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K., Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res. 2004 I 9; 32(3):936-48. See also Liu Y, Braasch D A, Nulf C J, Corey D R. Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 I 24; 43(7):1921-7. See also PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemical modification analysis, RNA 2003 September; 9(9):1034-48 and I U.S. Pat. Nos. 5,898,031 and 6107094 (Crooke) for production of modified/more stable siRNAs.

Several groups have described the development of DNA-based vectors capable of generating siRNA within cells. The method generally involves transcription of short hairpin RNAs that are efficiently processed to form siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. These reports describe methods to generate siRNAs capable of specifically targeting numerous endogenously and exogenously expressed genes.

Several studies have revealed that siRNA therapeutics are effective in viva in both mammals and in humans. Bitko et al., have shown that specific siRNA molecules directed against the respiratory syncytial virus (RSV) nucleocapsid N gene are effective in treating mice when administered intranasally (Bitko et al., Nat. Med. 2005, 11(1):50-55). For reviews of therapeutic applications of siRNAs see for example, Batik (Mol. Med. 2005, 83: 764-773); Chakraborty (Current Drug Targets 2007 8(3):469-82) and Dykxhoom, et al (Gene Therapy 2006, 13, 541-552). Furthermore, a phase I clinical study with short siRNA molecule that targets the VEGFR1 receptor for the treatment of Age-Related Macular Degeneration (AMD) has been conducted in human patients. In studies such siRNA administered by intravitreal (intraocular) injection was found effective and safe in 14 patients tested (Kaiser, Am J Ophthalmol. 2006 142(4):660-8).

The Nrf2 Gene and Polypeptide (gi|166295208|ref|NM_(—)006164.3| Homo sapiens Nuclear Factor (Erythroid-Derived 2)-like 2 (NFE2L2):

Nuclear factor erythroid-2 related factor 2 (Nrf2), a cap-and-collar basic leucine zipper transcription factor, positively regulates a transcriptional program that maintains cellular redox homeostasis and protects cells from oxidative insult, including insult from chemotherapeutic agents (Rangasamy T, et al. J Clin Invest 114, 1248 (2004)). Nrf2 activates transcription of its target genes through binding specifically to the antioxidant-response element (ARE) found in those gene promoters. The Nrf2-regulated transcriptional program includes a broad spectrum of genes, including antioxidants such as heme oxygenase-1, superoxide dismutase, glutathione reductase (GSR), glutathione peroxidase, thioredoxin, thioredoxin reductase, peroxiredoxins (PRDX).

Lung Cancer:

Lung cancer is a cancer that forms in tissues of the lung, usually in the cells lining air passages. The two main types are small cell lung cancer and non-small cell lung cancer. These types are diagnosed based on the morphology of the cells under a microscope. It is the most lethal of all cancers worldwide, responsible for up to 3 million deaths annually. In non-small cell lung cancer (NSCLC), results of standard treatment are poor except for the most localized cancers. Surgery is the most potentially curative therapeutic option for this disease; radiation therapy can produce a cure in a small number of patients and can provide palliation in most patients. Adjuvant chemotherapy may provide an additional benefit to patients with resected NSCLC. In advanced-stage disease, chemotherapy offers modest improvements in median survival, though overall survival is poor. Chemotherapy has produced short-term improvement in disease-related symptoms.

WO 2006/128041 discloses specific siRNA molecules for Nrf2 and its use for treating any cancer, preferably lung and kidney cancers. US20020164576 discloses a method of inhibiting tumor growth (preferably a lymphoma cancer) using antisense molecules directed to Nrf2 or specific antibodies. US20070042418 discloses the use of siRNA molecules for Nrf2 for treating cancer.

Despite the evident progress, there remains a continued need for improved molecules in particular improved siRNA compounds able to treat cancerous diseases, particularly lung cancers.

SUMMARY OF THE INVENTION

The invention provides novel double stranded oligoribonucleotides that inhibit the Nrf2 gene. The invention also provides a pharmaceutical composition comprising one or more such oligoribonucleotides, and a vector capable of expressing the oligoribonucleotide. The present invention also relates to methods and compositions for treating or preventing the incidence or severity of a cancerous disease, particularly various lung cancers. The methods and compositions involve administering to a mammal in need of such treatment a prophylactically or therapeutically effective amount of one or more compounds which down-regulate expression of the Nrf2 gene, particularly novel small interfering RNAs (siRNAs), small molecule inhibitors of Nrf2 as described herein or antibodies to Nrf2 polypeptide.

In certain embodiments the present invention provides a compound having the structure:

5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand) wherein each of N and N′ is a ribonucleotide which may be independently modified or unmodified in its sugar residue; wherein each of (N)_(x) and (N′)_(y) is an oligomer in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond; wherein each of x and y is an integer between 19 and 40; wherein each of Z and Z′ may be present or absent, but if present is 1-5 consecutive nucleotides covalently attached at the 3′ terminus of the strand in which it is present; and wherein the sequence of (N)_(x) is set forth as any one of SEQ ID NOS 504 to 1006 or 1507 to 2006, and the sequence of (N′)_(y) is set forth as the complementary sequence. The sequence of (N′)_(y) is a complementary sequence set forth as SEQ ID NOS: 1 to 503 or 1007 to 1506, respectively.

In some embodiments the covalent bond joining each consecutive Nor N′ is a phosphodiester bond. In various embodiments all the covalent bonds are phosphodiester bonds.

In various embodiments the compound comprises ribonucleotides wherein x=y and each of x and y is 19, 20, 21, 22 or 23. In some embodiments x=y=23. In other embodiments x=y=19.

In some embodiments the compound is blunt ended, for example wherein both Z and Z′ are absent. In an alternative embodiment, the compound comprises at least one 3′ overhang, wherein at least one of Z or Z′ is present. Z and Z′ can independently comprise one or more covalently linked modified or non-modified nucleotides, for example inverted dT or dA; dT, LNA, mirror nucleotide and the like. In some embodiments each of Z and V are independently selected from dT and dTdT.

In some embodiments Nor N′ comprises a modification in the sugar residue of one or more ribonucleotides. In other embodiments the compound comprises at least one ribonucleotide modified in the sugar residue. In some embodiments the compound comprises a modification at the 2′ position of the sugar residue. In some embodiments the modification in the 2′ position comprises the presence of an amino, a fluoro, an alkoxy or an alkyl moiety. In certain embodiments the 2′ modification comprises a methoxy moiety. A presently preferred modification is a 2′ methoxy of the sugar residue (2′-O-methyl; 2-O-Me; 2′-O—CH₃). In some embodiments the compound comprises modified alternating ribonucleotides in one or both of the antisense and the sense strands. In certain embodiments the compound comprises modified alternating ribonucleotides in the antisense and the sense strands. In other embodiments the compound comprises modified alternating ribonucleotides in the antisense strand only. In certain embodiments the middle ribonucleotide of the antisense strand is not modified; e.g. ribonucleotide in position 10 in a 19-mer strand or position 12 in a 23-mer strand.

In additional embodiments the compound comprises modified ribonucleotides in alternating positions wherein each N at the 5′ and 3′ termini of (N)_(x) are modified in their sugar residues, and each N′ at the 5′ and 3′ termini of (N′)_(y) are unmodified in their sugar residues. In some embodiments, neither (N)_(x) nor (N′)_(y) are phosphorylated at the 3′ and 5′ termini. In other embodiments either or both (N)_(x) and (N′)_(y) are phosphorylated at the 3′ termini. Preferably the siRNA molecule is either phosphorylated at the 3′ termini of both sense and anti-sense strands, or non-phosphorylated at all; similar results are obtained for both siRNAs. The siRNA molecules used in the in vitro experiments were phosphorylated at the 3′ termini of both sense and anti-sense strands while the siRNA molecules used in the in vivo experiments were non-phosphorylated at all.

More specifically, the present invention provides methods and compositions for treating a patient suffering from a cancerous disease, (e.g. lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer). In a particular embodiment, the cancer is lung cancer such as non-small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma. The methods of the invention comprising administering to the patient one or more compounds which down-regulate expression of the Nrf2 gene, particularly siRNAs that inhibit Nrf2, typically as a pharmaceutical composition, in a therapeutically effective dose so as to thereby treat the patient.

Still further, the invention relates to a method for treating or preventing the incidence or severity of a cancerous disease, particularly a lung disease in a patient comprising administering to the patient a composition comprising an effective amount of naked siRNA molecules. Preferably, the naked siRNA molecules are applied directly to lung for example via aerosol delivery which has the potential for delivering high concentrations of the therapeutic molecule to the inner lung.

Still further, the invention relates to the use of a therapeutically effective dose of an oligonucleotide for the preparation of a composition for treating a subject suffering from a cancerous disease, preferably lung cancer, wherein the oligonucleotide is set forth as any one of SEQ ID NOS:1-2006.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A549-C8 luciferase cells were transfected with non-targeting luciferase siRNA or Nrf2 siRNA-1 (sense strand SEQ ID NO: 10) at 20 nM concentration. Seventy two hours post transfection, cells were harvested and total RNA was isolated. Nrf2 knockdown as well as expression of two Nrf2 dependent target genes (GCLM and HO-1) were analyzed by real time RT-PCR.

FIG. 2: SCID-Beige mice injected i.v. with ARE-luciferase reporter tumor cells were inhaled with Nrf2 siRNA (sense strand SEQ ID NO: 10) twice during the 4th week of lung tumor growth. Control mice were inhaled with GFP siRNA. Mice were imaged before and after siRNA inhalation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to compounds which down-regulate expression of the Nrf2 gene particularly to novel small interfering RNAs (siRNAs), and to the use of these novel siRNAs in the treatment of various diseases and medical conditions in particular various forms of cancerous diseases. Such preferred siRNA compounds comprise sequences set forth as any one of SEQ ID NOS: 1 to 2006.

The inventors of the present invention have provided novel siRNAs to Nrf2 in order to treat any of the above diseases or disorders. Methods, molecules and compositions which inhibit Nrf2 are discussed herein at length, and any of said molecules and/or compositions may be beneficially employed in the treatment of a patient suffering from any of said conditions.

The present invention provides methods and compositions for inhibiting expression of a target Nrf2 gene in vivo. In general, the method includes administering oligoribonucleotides, such as small interfering RNAs (i.e., siRNAs) that are targeted to a particular Nrf2 mRNA and hybridize to, or interact with, the mRNAs under biological conditions (within the cell), or a nucleic acid material that can produce siRNA in a cell, in an amount sufficient to down-regulate expression of a target gene by an RNA interference mechanism. In particular, the subject method can be used to inhibit expression of the Nrf2 gene for treatment of a disease.

In accordance with the present invention, the siRNA molecules or inhibitors of the Nrf2 gene may be used as drugs to treat various pathologies accompanied by an elevated level of Nrf2 polypeptide.

The present invention provides double-stranded oligoribonucleotides (siRNAs), which down-regulate the expression of the Nrf2 gene. An siRNA of the invention is a duplex oligoribonucleotide in which the sense strand is derived from the mRNA sequence of the Nrf2 gene, and the antisense strand is complementary to the sense strand. In general, some deviation from the target mRNA sequence is tolerated without compromising the siRNA activity (see e.g. Czauderna et al 2003 Nucleic Acids Research 31(11), 2705-2716). An siRNA of the invention inhibits gene expression on a post-transcriptional level with or without destroying the mRNA. Without being bound by theory, siRNA may target the mRNA for specific cleavage and degradation and/or may inhibit translation from the targeted message.

As used herein, the term “Nrf2”, or “Nrf2 polypeptide” (gi|166295208|ref|NM_(—)006164.3| Homo sapiens nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) is defined as any homolog of the Nrf2 polypeptide having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to the Nrf2 gene, as either full-length or a fragment or a domain thereof, as a mutant or the polypeptide encoded by a spliced variant nucleic acid sequence, as a chimera with other polypeptides, provided that any of the above has the same or substantially the same biological function as the Nrf2 polypeptide.

Generally, the siRNAs used in the present invention comprise a ribonucleic acid comprising a double stranded structure, whereby the double-stranded structure comprises a first strand and a second strand, whereby the first strand comprises a first stretch of contiguous nucleotides and whereby said first stretch is at least partially complementary to a target nucleic acid, and the second strand comprises a second stretch of contiguous nucleotides and whereby said second stretch is at least partially identical to a target nucleic acid, whereby said first strand and/or said second strand comprises a plurality of groups of modified nucleotides having a modification at the 2′-position whereby within the strand each group of modified nucleotides is flanked on one or both sides by a flanking group of nucleotides whereby the flanking nucleotides forming the flanking group of nucleotides is either an unmodified nucleotide or a nucleotide having a modification different from the modification of the modified nucleotides. Further, said first strand and/or said second strand may comprise said plurality of modified nucleotides and may comprises said plurality of groups of modified nucleotides.

The group of modified nucleotides and/or the group of flanking nucleotides may comprise a number of nucleotides whereby the number is selected from the group comprising one nucleotide to 10 nucleotides. In connection with any ranges specified herein it is to be understood that each range discloses any individual integer between the respective figures used to define the range including said two figures defining said range. In the present case the group thus comprises one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides and ten nucleotides.

The pattern of modified nucleotides of said first strand may be shifted by one or more nucleotides relative to the pattern of modified nucleotides of the second strand.

The modifications discussed above may be selected from the group comprising amino, fluoro, methoxy alkoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃, OCN, O—, S—, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃; heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.

The double stranded structure of the siRNA may be blunt ended, on one or both sides. More specifically, the double stranded structure may be blunt ended on the double stranded structure's side which is defined by the 5′-end of the first strand and the 3′-end of the second strand, or the double stranded structure may be blunt ended on the double stranded structure's side which is defined by at the 3′-end of the first strand and the 5′-end of the second strand. Additionally, at least one of the two strands may have an overhang of at least one nucleotide at the 5′-end; the overhang may consist of at least one deoxyribonucleotide. At least one of the strands may also optionally have an overhang of at least one nucleotide at the 3′-end.

The length of the double-stranded structure of the siRNA is typically from about 17 to 21 and more preferably 18 or 19 bases. Further, the length of said first strand and/or the length of said second strand may independently from each other be selected from the group comprising the ranges of from about 15 to about 23 bases, 17 to 21 bases and 18 or 19 bases.

Additionally, the complementarily between said first strand and the target nucleic acid may be perfect, or the duplex formed between the first strand and the target nucleic acid may comprise at least 15 nucleotides wherein there is one mismatch or two mismatches between said first strand and the target nucleic acid forming said double-stranded structure.

Substantially complementary refers to complementarity of greater than about 84%, to another sequence. For example in a duplex region consisting of 19 base pairs one mismatch results in 94.7% complementarity, two mismatches results in about 89.5% complementarity and 3 mismatches results in about 84.2% complementarity, rendering the duplex region substantially complementary. Accordingly substantially identical refers to identity of greater than about 84%, compared to another sequence.

In some cases both the first strand and the second strand each comprise at least one group of modified nucleotides and at least one flanking group of nucleotides, whereby each group of modified nucleotides comprises at least one nucleotide and whereby each flanking group of nucleotides comprising at least one nucleotide with each group of modified nucleotides of the first strand being aligned with a flanking group of nucleotides on the second strand, whereby the most terminal 5′ nucleotide of the first strand is a nucleotide of the group of modified nucleotides, and the most terminal 3′ nucleotide of the second strand is a nucleotide of the flanking group of nucleotides. Each group of modified nucleotides may consist of a single nucleotide and/or each flanking group of nucleotides may consist of a single nucleotide.

Additionally, it is possible that on the first strand the nucleotide forming the flanking group of nucleotides is an unmodified nucleotide which is arranged in a 3′ direction relative to the nucleotide forming the group of modified nucleotides, and on the second strand the nucleotide forming the group of modified nucleotides is a modified nucleotide which is arranged in 5′ direction relative to the nucleotide forming the flanking group of nucleotides.

Further the first strand of the siRNA may comprise eight to twelve, preferably nine to eleven, groups of modified nucleotides, and the second strand may comprise seven to eleven, preferably eight to ten, groups of modified nucleotides.

The first strand and the second strand may be linked by a loop structure, which may be comprised of a non-nucleic acid polymer such as, inter alia, polyethylene glycol. Alternatively, the loop structure may be comprised of a nucleic acid.

Further, the 5′-terminus of the first strand of the siRNA may be linked to the 3′-terminus of the second strand, or the 3′-end of the first strand may be linked to the 5′-terminus of the second strand, said linkage being via a nucleic acid linker typically having a length between 10-2000 nucleobases.

In particular, the invention provides a compound having structure A:

5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)

-   -   wherein each N and N′ is a ribonucleotide which may         independently be modified or unmodified in its sugar residue and         each of (N)_(x) and (N′)_(y) is oligomer in which each         consecutive N or N′ is joined to the next N or N′ by a covalent         bond;     -   wherein each of x and y is an integer between 19 and 40;     -   wherein each of Z and Z′ may be present or absent, but if         present is dTdT and is covalently attached at the 3′ terminus of         the strand in which it is present;     -   and wherein the sequence of (N)_(x) is set forth as any one of         SEQ ID NOS 504 to 1006 or 1507 to 2006, and the sequence of         (N′)_(y) is set forth as the complementary sequence. The         sequence of (N′)_(y) is a complementary sequence set forth as         SEQ ID NOS: 1 to 503 or 1007 to 1506, respectively.

It will be readily understood by those skilled in the art that the compounds of the present invention consist of a plurality of nucleotides, which are linked through covalent linkages. Each such covalent linkage may be a phosphodiester linkage, a phosphothioate linkage, or a combination of both, along the length of the nucleotide sequence of the individual strand. Other possible backbone modifications are described inter alfa in U.S. Pat. Nos. 5,587,361; 6,242,589; 6,277,967; 6,326,358; 5,399,676; 5,489,677; and 5,596,086.

In particular embodiments, x and y are preferably an integer between about 19 to about 27, most preferably from about 19 to about 23. In a particular embodiment of the compound of the invention, x may be equal to y (viz., x=y) and in preferred embodiments x=y=19 or x=y=21. In a particularly preferred embodiment x=y=19.

In one embodiment of the compound of the invention, Z and Z′ are both absent; in another embodiment one of Z or Z′ is present.

In one embodiment of the compound of the invention, all of the ribonucleotides of the compound are unmodified in their sugar residues.

In preferred embodiments of the compound of the invention, at least one ribonucleotide is modified in its sugar residue, preferably a modification at the 2′ position. The modification at the 2′ position results in the presence of a moiety which is preferably selected from the group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. In a presently most preferred embodiment the moiety at the 2′ position is methoxy (2′-0-methyl).

In preferred embodiments of the invention, alternating ribonucleotides are modified in both the antisense and the sense strands of the compound. In particular the siRNA used in the Examples has been such modified such that a 2′ O-Me group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i.e. a 2′-O-Me group was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand. Additionally, it is to be noted that the in case of these particular nucleic acids according to the present invention the first stretch is identical to the first strand and the second stretch is identical to the second strand and these nucleic acids are also blunt ended.

According to one preferred embodiment of the invention, the antisense and the sense strands of the siRNA molecule are both phosphorylated only at the 3′-terminus and not at the 5′-terminus. According to another preferred embodiment of the invention, the antisense and the sense strands are both non-phosphorylated both at the 3′-terminus and also at the 5′-terminus. According to yet another preferred embodiment of the invention, the 1^(st) nucleotide in the 5′ position in the sense strand is specifically modified to abolish any possibility of in vivo 5′-phosphorylation.

In another embodiment of the compound of the invention, the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues.

The invention further provides a vector capable of expressing any of the aforementioned oligoribonucleotides in unmodified form in a cell after which appropriate modification may be made.

The invention also provides a composition comprising one or more of the compounds of the invention in a carrier, preferably a pharmaceutically acceptable carrier. This composition may comprise a mixture of two or more different siRNAs.

The invention also provides a composition which comprises the above compound of the invention covalently or non-covalently bound to one or more compounds of the invention in an amount effective to inhibit Nrf2 and a carrier. This composition may be processed intracellularly by endogenous cellular complexes to produce one or more oligoribonucleotides of the invention.

The invention also provides a composition comprising a carrier and one or more of the compounds of the invention in an amount effective to down-regulate expression in a cell of Nrf2, which compound comprises a sequence substantially complementary to the sequence of (N)_(x).

Additionally the invention provides a method of down-regulating the expression of Nrf2 by at least 50% as compared to a control comprising contacting an mRNA transcript of Nrf2 with one or more of the compounds of the invention.

In one embodiment the oligoribonucleotide is down-regulating Nrf2, whereby the down-regulation of Nrf2 is selected from the group comprising down-regulation of gene function, down-regulation of polypeptide and down-regulation of mRNA expression.

In one embodiment the compound is down-regulating the Nrf2 polypeptide, whereby the down-regulation is selected from the group comprising down-regulation of function (which may be examined by an enzymatic assay or a binding assay with a known interactor of the native gene/polypeptide, inter alia), down-regulation of protein (which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia) and down-regulation of mRNA expression (which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia).

The invention also provides a method of treating a patient suffering from a disease accompanied by an elevated level of Nrf2, the method comprising administering to the patient a composition of the invention in a therapeutically effective dose thereby treating the patient.

More particularly, the invention provides an oligoribonucleotide wherein one strand comprises consecutive nucleotides having, from 5′ to 3′, the sequence set forth in Table A (19mer siRNA molecules, SEQ ID NOS: 1-1006) or in Table B (21mer siRNA molecules, SEQ ID NOS: 1007-2006), or a homolog thereof wherein in up to 2 of the nucleotides in each terminal region a base is altered. Preferred 19mer siRNA molecules comprise the sense and corresponding antisense sequences listed in Table C. More preferred siRNA comprises SEQ ID NO: 10 (sense sequence) and SEQ ID NO: 513 (antisense sequence). The terminal region of the oligonucleotide refers to bases 1-4 and/or 16-19 in the 19-mer sequence and to bases 1-4 and/or 18-21 in the 21-mer sequence.

The presently most preferred compound of the invention is a blunt-ended 19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent. The oligonucleotide molecule is either phosphorylated at 3′ termini of both sense and anti-sense strands, or non-phosphorylated at all; or having I″ nucleotide in the 5′ position on the sense strand specifically modified to abolish any possibility of in vivo 5′-phosphorylation. The alternating ribonucleotides are modified at the 2′ position in both the antisense and the sense strands, wherein the moiety at the 2′ position is methoxy (2′-0-methyl) and wherein the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues.

In certain embodiments the present invention provides a compound having the structure

5′ (N)x 3′ antisense strand 3′ (N′)y 5′ sense strand wherein each of x and y=19 and (N)_(x) and (N′)_(y) are fully complementary; wherein alternating ribonucleotides in (N)x and (N′)y are modified to result in a 2′-O-methyl modification in the sugar residue of the ribonucleotides; wherein each N at the 5′ and 3′ termini of (N)_(x) are modified; wherein each N′ at the 5′ and 3′ termini of (N′)_(y) are unmodified; wherein each of (N)_(x) and (N′)_(y) is selected from the group of oligomers set forth in any one of Tables A and B (SEQ ID NOS: 1-2006). (N)_(x) and (N′)_(y) may be phosphorylated or non-phosphorylated at the 3′ and 5′ termini.

In certain embodiments of the invention, alternating ribonucleotides are modified in the 2′ position of the sugar residue in both the antisense and the sense strands of the compound. In particular the siRNAs may be modified such that a 2′-O-methyl (Me) group is present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i.e. a 2′-O-Me group, is present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand. These particular siRNA compounds may also be blunt ended.

In certain embodiments of the compounds of the invention having alternating ribonucleotides modified in one or both of the antisense and the sense strands of the compound; for 19-mers and 23-mers the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues. For 21-mers the ribonucleotides at the 5′ and 3′ termini of the sense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the antisense strand are unmodified in their sugar residues. As mentioned above, it is preferred that the middle nucleotide of the antisense strand is unmodified.

In some preferred embodiments the middle ribonucleotide in the first strand (antisense) is an unmodified nucleotide. For example, in a 19-oligomer antisense strand, ribonucleotide number 10 is unmodified; in a 21-oligomer antisense strand, ribonucleotide number 11 is unmodified; and in a 23-oligomer antisense strand, ribonucleotide number 12 is unmodified. The modifications or pattern of modification, if any, of the siRNA must be planned to allow for this.

Delivery: The siRNA molecules of the present invention may be delivered to the target tissue (such as the lung) by direct application of the naked molecules admixed with a carrier or a diluent using an aerosol.

For administration via the upper respiratory tract, the composition is formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, at an appropriate concentration for oro-nasal administration as an aerosol. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's Pharmaceutical Sciences 16th edition, Ed. Arthur Osol, page 1445 (1980)). One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and/or upper respiratory administration.

Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

The compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no auxiliary agents or substances are present that might affect or mediate uptake of nucleic acid in the cells of the lungs.

Aerosol dosage, formulations and delivery systems may be selected for a particular therapeutic application, as described, for example, in Gonda, I. “Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract,” in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313, 1990; and in Moren, “Aerosol dosage forms and formulations,” in: Aerosols in Medicine, Principles, Diagnosis and Therapy, Moren, et al., Eds. Elsevier, Amsterdam, 1985. The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultrasonication or high pressure treatment.

Various suitable devices and methods of inhalation which can be used to administer particles to a patient's respiratory tract are known in the art. Nebulizers create a fine mist from a solution or suspension, which is inhaled by the patient. The devices described in U.S. Pat. No. 5,709,202 to Lloyd, et al., can be used. An MDI typically includes a pressurized canister having a meter valve, wherein the canister is filled with the solution or suspension and a propellant. The solvent itself may function as the propellant, or the composition may be combined with a propellant, such as freon. The composition is a fine mist when released from the canister due to the release in pressure. The propellant and solvent may wholly or partially evaporate due to the decrease in pressure.

The term “naked siRNA” refers to siRNA molecules that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. For example, siRNA in PBS is “naked siRNA”. However, the siRNA molecules of the invention can also be delivered in liposome formulations and lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, see, for example, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recently been successfully used for inhibition in primates; for further details see Tolentino et al., Retina 24(1) February 2004 I 132-138. Respiratory formulations for siRNA are described in U.S. patent application No. 2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and other steroid and lipid conjugated siRNAs) can been used for delivery see Soutschek et al Nature 432: 173-177 (2004); and Lorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004).

The siRNAs or pharmaceutical compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.

The “therapeutically effective dose” for purposes herein is thus determined by such considerations as are known in the art. The dose must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. The compounds of the present invention can be administered by any of the conventional routes of administration. It should be noted that the compound can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful. Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parental routes of administration. The liquid compositions include aqueous solutions, with and without organic co-solvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles. In addition, under certain circumstances the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration. The patient being treated is a warm-blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention and they include liposomes and microspheres. Examples of delivery systems useful in the present invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. In one specific embodiment of this invention topical and transdermal formulations are particularly preferred.

In general, the active dose of compound for humans is in the range of from 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of one dose per day or twice or three or more times per day for a period of 1-4 weeks or longer.

The term “treatment” as used herein refers to administration of a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring. Thus “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a disease in particular a cancerous disease such as lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer. In a particular embodiment, the cancer is lung cancer such as non-small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma

In another aspect of the invention a pharmaceutical composition is provided which comprises any of the above oligoribonucleotides or vectors and a pharmaceutically acceptable carrier. Another aspect of the invention is the use of a therapeutically effective amount of any of the above oligoribonucleotides or vectors for the preparation of a medicament for treating a patient suffering from a cancer disease.

By “cancerous disease” is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Examples of cancerous diseases include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio sarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyo sarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, crailiopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwamioma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

In one embodiment, the present invention provides a pharmaceutical composition comprising an Nrf2 inhibitory molecule, preferable an siRNA molecule that decreases the expression of the Nrf2 gene in combination with a chemotherapeutic agent. Without being bound by theory, since Nrf2 positively regulates drug detoxification enzymes, targeting this molecule may have a broad effect on all anticancer drugs. In various embodiments, the Nrf2 inhibitory molecule is administered prior to, concurrently with, or following administration of a chemotherapeutic. Without wishing to be bound by theory, administration of an Nrf2 inhibitory molecule likely enhances the accumulation or efficacy of a chemotherapeutic agent.

Compositions and methods of the invention may be used in combination with any conventional therapy known in the art. In one embodiment, the Nrf2 inhibitory molecules of the invention may be used in combination with one or more anti-cancerous therapy known in the art. Thus the Nrf2 inhibitors may be used before, during or following anti-cancerous therapy Exemplary anti-cancerous therapies include, for example, chemotherapy, cryotherapy, hormone therapy, growth factor inhibitors, radiotherapy, and surgery. Chemotherapeutics typically used in the treatment of a cancerous disease are for example abiraterone acetate, altretamine, anhydrovinbiastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide, cachectin, cemadotin, chiorambucil, cyclophosphamide, 3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNTJ), cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, oxaliplatin paclitaxel, prednimustine, procarbazine, RPR1 09881, satrapaltin, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinbiastine, vincristine, vindesine sulfate, and vinflunine. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Heliman (editors), 6th edition (Fel. 15, 2001), Lippincott Williams & Wilkins Publishers. A preferred combination according to the present invention is an siRNA molecule targeting Nrf2 with platinum drugs, more preferably an siRNA molecule set forth in Table C with a platinum drug, preferably carboplatin. Without being bound by theory, the Nrf2 siRNA inhibitors of the invention are found to be efficient promoters for the antineoplastic potential of platinum drugs, causing additive/synergistic effects in cancer cells. Platinum drugs include carboplatin, cisplatin, oxaliplatin and satrapaltin inter alia; see Kelland and Farrell, Platinum-based drugs in cancer therapy (Cancer drug discovery & development) Lavoisier 2000 which is hereby incorporated by reference.

The compounds which reduce or prevent the cancerous disease such as lung cancer, eg the novel siRNAs inter alia are preferably administered directly to the inner lung as naked siRNA in a vehicle such as PBS or other physiological solutions, but may alternatively be administered with a delivery vehicle as described above.

The present invention also provides for a process of preparing a pharmaceutical composition, which comprises:

-   -   obtaining one or more double stranded compound of the invention;         and     -   admixing said compound with a pharmaceutically acceptable         carrier.

The present invention also provides for a process of preparing a pharmaceutical composition, which comprises admixing one or more compounds of the present invention with a pharmaceutically acceptable carrier.

In a preferred embodiment, the compound used in the preparation of a pharmaceutical composition is admixed with a carrier in a pharmaceutically effective dose. In a particular embodiment the compound of the present invention is conjugated to a steroid or to a lipid or to another suitable molecule e.g. to cholesterol.

Modifications or analogs of nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.

Accordingly, the present invention also includes all analogs of, or modifications to, a oligonucleotide of the invention that does not substantially affect the function of the polynucleotide or oligonucleotide. In a preferred embodiment such modification is related to the base moiety of the nucleotide, to the sugar moiety of the nucleotide and/or to the phosphate moiety of the nucleotide.

In embodiments of the invention, the nucleotides can be selected from naturally occurring or synthetically modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of the oligonucleotides include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl-adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenine, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanine, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanine, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

In addition, analogs of nucleotides can be prepared wherein the structures of the nucleotides are fundamentally altered and are better suited as therapeutic or experimental reagents. An example of a nucleotide analog is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. Further, PNAs have been shown to bind more strongly to a complementary DNA sequence than to a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand. Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.

In one embodiment the modification is a modification of the phosphate moiety, whereby the modified phosphate moiety is selected from the group comprising phosphothioate.

The compounds of the present invention can be synthesized by any of the methods that are well-known in the art for synthesis of ribonucleic (or deoxyribonucleic) oligonucleotides. Such synthesis is, among others, described in Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313; the synthesis of thioates is, among others, described in Eckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules is described in Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 and respective downstream processes are, among others, described in Pingoud A. et. al., in IRL Press 1989 edited by Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 edited by Herdewijn P.; Kap. 2: 17-31 (supra).

Other synthetic procedures are known in the art e.g. the procedures as described in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684; and Wincott et al., 1997, Methods Mol. Bio., 74, 59, and these procedures may make use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. The modified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotides are incorporated as desired.

The oligonucleotides of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.

It is noted that a commercially available machine (available, inter alia, from Applied Biosystems) can be used; the oligonucleotides are prepared according to the sequences disclosed herein. Overlapping pairs of chemically synthesized fragments can be ligated using methods well known in the art (e.g., see U.S. Pat. No. 6,121,426). The strands are synthesized separately and then are annealed to each other in the tube. Then, the double-stranded siRNAs are separated from the single-stranded oligonucleotides that were not annealed (e.g. because of the excess of one of them) by HPLC. In relation to the siRNAs or siRNA fragments of the present invention, two or more such sequences can be synthesized and linked together for use in the present invention.

The compounds of the invention can also be synthesized via a tandem synthesis methodology, as described in US patent application publication No. US2004/0019001 (McSwiggen), wherein both siRNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siRNA fragments or strands that hybridize and permit purification of the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker.

The present invention further provides for a pharmaceutical composition comprising two or more siRNA molecules for the treatment of any of the diseases and conditions mentioned herein, whereby said two molecules may be physically mixed together in the pharmaceutical composition in amounts which generate equal or otherwise beneficial activity, or may be covalently or non-covalently bound, or joined together by a nucleic acid linker of a length ranging from 2-100, preferably 2-50 or 2-30 nucleotides. In one embodiment, the siRNA molecules are comprised of a double-stranded nucleic acid structure as described herein, wherein the two siRNA sequences are selected from Tables A or B.

In another embodiment, the siRNA molecules are comprised of a double-stranded nucleic acid structure, wherein the first siRNA sequence is selected from Tables A or B, and the second siRNA molecule targets another anti-cancer gene, thereby providing beneficial activity. The tandem double-stranded structure which comprises two or more siRNA sequences is processed intracellularly to form two or more different siRNAs. Such second siRNA molecule is preferably an siRNA molecule that targets a anti-cancer gene. Preferred anti-cancer genes are for example gene encoding growth factors such as insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet derived growth factor (PDGF).

The siRNA molecules are covalently or non-covalently bound or joined by a linker to form a tandem siRNA molecule. Such tandem siRNA molecules comprising two siRNA sequences are typically of 38-150 nucleotides in length, more preferably 38 or 40-60 nucleotides in length, and longer accordingly if more than two siRNA sequences are included in the tandem molecule. A longer tandem molecule comprised of two or more longer sequences which encode siRNA produced via internal cellular processing, e.g., long dsRNAs, is also envisaged, as is a tandem molecule encoding two or more shRNAs. Such tandem molecules are also considered to be a part of the present invention.

siRNA molecules that target Nrf2 may be the main active component in a pharmaceutical composition, or may be one active component of a pharmaceutical composition containing two or more siRNAs (or molecules which encode or endogenously produce two or more siRNAs, be it a mixture of molecules or one or more tandem molecules which encode two or more siRNAs), said pharmaceutical composition further being comprised of one or more additional siRNA molecule which targets one or more additional gene. Simultaneous inhibition of Nrf2 and said additional gene(s) will likely have an additive or synergistic effect for treatment of the diseases disclosed herein.

In a preferred embodiment, the one or more additional siRNA molecules target an anti-cancer gene, thus having an additive or synergistic effect with the Nrf2 siRNA. The additional siRNA molecules may target one or more of the anti-cancer genes defined above.

As disclosed herein, aptamers may also be used in the present invention alone or in combination with the novel siRNAs disclosed herein for targeting NRF2 and for the treatment of any one of the conditions disclosed herein. For example, an aptamer can be used with any one of the siRNAs disclosed herein in combination therapy for the treatment of any one of the conditions disclosed herein. The novel pharmaceutical composition employed for such a combination therapy, which is also part of the present invention, may comprise an siRNA of the present invention covalently or non-covalently attached to an aptamer. Aptamers are RNA or DNA single-strand or double-strand oligonucleic acids which bind to a target protein and do not generally exhibit non-specific effects. Aptamers can be modified for stability or other desired qualities in accordance with any nucleic acid modifications disclosed herein and/or known to one of skill in the art. Modifications to aptamers can be introduced anywhere in the molecule, such as the 5′ or 3′ termini, or at any internally defined modification site. For example, RNA aptamers can be stabilized with 2′-fluoro or 2′-amino modified pyrimidines. Aptamers can also be linked to reporter molecules or linker chemistries and can be attached to beads or other solid support if necessary (e.g., 5′ or 3′ amino, thiol ester or biotin groups). Thioaptamers are aptamers which contain sulfur modifications at specific internucleoside phosphoryl sites, and may possess enhanced stability, nuclease resistance, target affinity and/or selectivity. Examples of thioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate (S2-ODN) oligodeoxy thioaptamers. For further information on aptamers and thioaptamers see U.S. Pat. Nos. 5,218,088 and 6,423,493.

Additionally, the siRNA disclosed herein or any nucleic acid molecule comprising or encoding such siRNA can be linked or bound (covalently or non-covalently) to antibodies (including aptamer molecules) against cell surface internalizable molecules expressed on the target cells, in order to achieve enhanced targeting for treatment of the diseases disclosed herein. For example, anti-Fas antibody (preferably a neutralizing antibody) may be combined (covalently or non-covalently) with a Nrf2 siRNA molecule. In another example, an aptamer which can act like a ligand/antibody may be combined (covalently or non-covalently) with a Nrf2 siRNA molecule.

The term “Covalent bonding” as used herein refers to chemical bonding that is characterized by the sharing of pairs of electrons between atoms.

The term “Noncovalent bonding” as used herein refers to a variety of interactions that are not covalent in nature between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together, usually in a specific orientation or conformation. These noncovalent interactions include: ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces anddipole-dipole bonds.

The compounds of the present invention can be delivered either directly or with viral or non-viral vectors. When delivered directly the sequences are generally rendered nuclease resistant. Alternatively the sequences can be incorporated into expression cassettes or constructs such that the sequence is expressed in the cell as discussed herein below. Generally the construct contains the proper regulatory sequence or promoter to allow the sequence to be expressed in the targeted cell. Vectors optionally used for delivery of the compounds of the present invention are commercially available, and may be modified for the purpose of delivery of the compounds of the present invention by methods known to one of skill in the art.

It is also envisaged that a long oligonucleotide (typically 25-500 nucleotides in length) comprising one or more stem and loop structures, where stern regions comprise the sequences of the oligonucleotides of the invention, may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes (e.g. by DROSHA and DICER as described above) to produce one or more smaller double stranded oligonucleotides (siRNAs) which are oligonucleotides of the invention. This oligonucleotide can be termed a tandem shRNA construct. It is envisaged that this long oligonucleotide is a single stranded oligonucleotide comprising one or more stem and loop structures, wherein each stem region comprises a sense and corresponding antisense siRNA sequence of an NRF2 gene. In particular, it is envisaged that this oligonucleotide comprises sense and antisense siRNA sequences as depicted as any one of SEQ ID NO: 1-2006.

As used herein, the term “inhibition” of the Nrf2 gene means inhibition of the gene expression (transcription or translation) or polypeptide activity.

Although the inhibitor may be an siRNA molecule, other inhibitors contemplated to be used in the methods of the invention to inhibit the Nrf2 gene and to treat the diseases and conditions described herein are inter alia antibodies, preferably neutralizing antibodies or fragments thereof, including single chain antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, aptamers, proteins, polypeptides and peptides including peptidomimetics and dominant negatives, and also expression vectors expressing all the above. Additional inhibitors may be small chemical molecules, which generally have a molecular weight of less than 2000 daltons, more preferably less than 1000 daltons, even more preferably less than 500 daltons. These inhibitors may act as follows: small molecules may affect expression and/or activity; antibodies may affect activity; all kinds of antisense may affect the gene expression; and dominant negative polypeptides and peptidomimetics may affect activity; expression vectors may be used inter alia for delivery of antisense or dominant-negative polypeptides or antibodies.

The term “antibody” refers to IgG, IgM, IgD, IgA, and IgE antibody, inter alia. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of antibodies comprising an antigen-binding domain, e.g. antibodies without the Fc portion, single chain antibodies, miniantibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc. The term “antibody” may also refer to antibodies against polynucleotide sequences obtained by cDNA vaccination. The term also encompasses antibody fragments which retain the ability to selectively bind with their antigen or receptor and are exemplified as follows, inter alia:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule which can be produced by digestion of whole antibody with the enzyme papain to yield a light chain and a portion of the heavy chain; (2) (Fab′)₂, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′₂) is a dimer of two Fab fragments held together by two disulfide bonds; (3) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (4) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Antisense Molecules

By the term “antisense” (AS) or “antisense fragment” is meant a polynucleotide fragment (comprising either deoxyribonucleotides, ribonucleotides or a mixture of both) having inhibitory antisense activity, said activity causing a decrease in the expression of the endogenous genomic copy of the corresponding gene. An AS polynucleotide is a polynucleotide which comprises consecutive nucleotides having a sequence of sufficient length and homology to a sequence present within the sequence of the target gene to permit hybridization of the AS to the gene. Many reviews have covered the main aspects of antisense (AS) technology and its enormous therapeutic potential (Aboul-Fadl T., Curr Med. Chem. 2005; 12(19):2193-214; Crooke S T, Curr Mol. Med. 2004 August; 4(5):465-87; Crooke S T, Annu Rev Med. 2004; 55:61-95; Vacek M et al., Cell Mol Life Sci. 2003 May; 60(5):825-33; Cho-Chung Y S, Arch Pharm Res. 2003 March; 26(3):183-91. There are further reviews on the chemical (Crooke, 1995; Uhlmann et al, 1990), cellular (Wagner, 1994) and therapeutic (Hanania, et al, 1995; Scanlon, et al, 1995; Gewirtz, 1993) aspects of this technology. Antisense intervention in the expression of specific genes can be achieved by the use of synthetic AS oligonucleotide sequences (for recent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; LevLehman et al, 1997).

AS oligonucleotide sequences may be short sequences of DNA, typically 15-30 mer but may be as small as 7 mer (Wagner et al, 1996), designed to complement a target mRNA of interest and form an RNA:AS duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant in RNA. Moreover, certain AS nucleotide sequences can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation (Calabretta et al, 1996 Semin Oncol. 23(1):78-87). In that case, RNase H will cleave the RNA component of the duplex and can potentially release the AS to further hybridize with additional molecules of the target RNA. An additional mode of action results from the interaction of AS with genomic DNA to form a triple helix which can be transcriptionally inactive.

The sequence target segment for the antisense oligonucleotide is selected such that the sequence exhibits suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and shows a low potential for self-dimerization or self-complementation [Anazodo et al., 19961. For example, the computer program OLIGO (Primer Analysis Software, Version 3.4), can be used to determine antisense sequence melting temperature, free energy properties, and to estimate potential self-dimer formation and self-complimentary properties. The program allows the determination of a qualitative estimation of these two parameters (potential self-dimer formation and self-complimentary) and provides an indication of “no potential” or “some potential” or “essentially complete potential”. Using this program target segments are generally selected that have estimates of no potential in these parameters. However, segments can be used that have “some potential” in one of the categories. A balance of the parameters is used in the selection as is known in the art. Further, the oligonucleotides are also selected as needed so that analogue substitution do not substantially affect function.

Phosphorothioate antisense oligonucleotides do not normally show significant toxicity at concentrations that are effective and exhibit sufficient pharmacodynamic half-lives in animals (Agarwal et al., 1996) and are nuclease resistant. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), for the establishment of tectal plate formation in chick (Galileo et al., 1991) and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (ephithelial vs. neuroblastic cells, which differ in their colony forming abilities, tumorigenicity and adherence) (Rosolen et al., 1990; Whitesell et al, 1991). Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells (Morrison, 1991) in a saturable and specific manner. Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes (Akhter et al., 1991). Following their interaction with the cellular plasma membrane, they are actively (or passively) transported into living cells (Loke et al., 1989), in a saturable mechanism predicted to involve specific receptors (Yakubov et al., 1989).

Ribozymes

A “ribozyme” is an RNA molecule that possesses RNA catalytic ability (see Cech for review) and cleaves a specific site in a target RNA. In accordance with the present invention, ribozymes which cleave mRNA may be utilized as inhibitors. This may be necessary in cases where antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then be used that will target the a gene associated with a bone marrow disease. The number of RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry. (Hampel and Tritz, 1989; Uhlenbeck, 1987).

Ribozymes catalyze the phosphodiester bond cleavage of RNA. Several ribozyme structural families have been identified including Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Pat. No. 5,225,347). The latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994). In general the ribozyme has a length of from about 30-100 nucleotides. Delivery of ribozymes is similar to that of AS fragments and/or siRNA molecules.

Screening of Inactivation Compounds for Nrf2:

Some of the compounds and compositions of the present invention may be used in a screening assay for identifying and isolating compounds that modulate the activity of the NRF2 gene, in particular compounds that modulate a disorder accompanied by an elevated level of NRF2. The compounds to be screened comprise inter alia substances such as small chemical molecules and antisense oligonucleotides.

The inhibitory activity of the compounds of the present invention on Nrf2 expression may be used to determine the interaction of an additional compound with the target polypeptide, e.g., if the additional compound competes with the oligonucleotides of the present invention for inhibition of Nrf2, or if the additional compound rescues said inhibition. The inhibition or activation can be tested by various means, such as, inter alia, assaying for the product of the activity of the Nrf2 polypeptide or displacement of binding compound from the Nrf2 polypeptide in radioactive or fluorescent competition assays.

The present invention is illustrated in detail below with reference to the Examples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.

EXAMPLES General Methods in Molecular Biology

Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and as in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and as in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, Calif. (1990). In situ (In cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al., 1996, Blood 87:3822.) Methods of performing RT-PCR are also well known in the art.

Example 1 Generation of Sequences for Active siRNA Compounds

Using proprietary algorithms and the known sequence of Nrf2 (gi2014957), the sequences of many potential siRNAs were generated. Table A shows a list of 19-mers siRNAs specific to Nrf2 which are either human-specific, or human and cross-species with other species. Table B shows a list of 21-mers siRNAs specific to Nrf2 which are either human-specific, or human and cross-species with other species. All siRNAs are depicted in 5′ to 3′ orientation, and the sense and complementary antisense sequences are depicted on the same line in the Table. The sense siRNAs in Table A have SEQ ID NOS: 1-503 and the antisense siRNAs in Table A have SEQ ID NOS: 504-1006. The sense siRNAs in Table B have SEQ ID NOS: 1007-1506 and the antisense siRNAs in Table B have SEQ ID NOS: 1507-2006.

TABLE A 19-mers siRNAs specific to Nrf2 (SEQ ID NOS: 1-1006) Sense siRNA Antisense siRNA Human-20149575 1 GGGGUAAGAAUAAAGUGGC GCCACUUUAUUCUUACCCC [1624-1642](19/19) 2 GGAGGGGUAAGAAUAAAGU ACUUUAUUCUUACCCCUCC [1621-1639](19/19) 3 GCAAAACUAACCACUAUGU ACAUAGUGGUUAGUUUUGC [2270-2288](19/19) 4 AAGGAGAAAAUGACAAAAG CUUUUGUCAUUUUCUCCUU [1741-1759](19/19) 5 CAGAAUUGCAGAAAAAGAA UUCUUUUUCUGCAAUUCUG [1647-1665](19/19) 6 GGGUAAGAAUAAAGUGGCU AGCCACUUUAUUCUUACCC [1625-1643](19/19) 7 GCCCUCACCUGCUACUUUA UAAAGUAGCAGGUGAGGGC [1010-1028](19/19) 8 AGGGGUAAGAAUAAAGUGG CCACUUUAUUCUUACCCCU [1623-1641](19/19) 9 AGGAGGGGUAAGAAUAAAG CUUUAUUCUUACCCCUCCU [1620-1638](19/19) 10 UCCCGUUUGUAGAUGACAA UUGUCAUCUACAAACGGGA [493-511](19/19) 11 AGGAGAAAAUGACAAAAGC GCUUUUGUCAUUUUCUCCU [1742-1760](19/19) 12 GCUCAGAAUUGCAGAAAAA UUUUUCUGCAAUUCUGAGC [1644-1662](19/19) 13 GAGAAAGAAUUGCCUGUAA UUACAGGCAAUUCUUUCUC [1392-1410](19/19) 14 UCCGGCAUUUCACUAAACA UGUUUAGUGAAAUGCCGGA [1143-1161](19/19) 15 GAGUUACAGUGUCUUAAUA UAUUAAGACACUGUAACUC [699-717](19/19) 16 GCCAAAACUAGUAUAGAAA UUUCUAUACUAGUUUUGGC [2044-2062](19/19) 17 AGCCAGAUGUUAAGAAAAA UUUUUCUUAACAUCUGGCU [1909-1927](19/19) 18 GAAGCCAGAUGUUAAGAAA UUUCUUAACAUCUGGCUUC [1907-1925](19/19) 19 AGAAGCCAGAUGUUAAGAA UUCUUAACAUCUGGCUUCU [1906-1924](19/19) 20 UCAGAAUUGCAGAAAAAGA UCUUUUUCUGCAAUUCUGA [1646-1664](19/19) 21 UAGGAGGGGUAAGAAUAAA UUUAUUCUUACCCCUCCUA [1619-1637](19/19) 22 CACUCUCUGAACUUCUAAA UUUAGAAGUUCAGAGAGUG [1036-1054](19/19) 23 UAGCCCCUGUUGAUUUAGA UCUAAAUCAACAGGGGCUA [628-646](19/19) 24 GAUCUGCCAACUACUCCCA UGGGAGUAGUUGGCAGAUC [406-424](19/19) 25 GCAAAAUCAUAGCCAAAAC GUUUUGGCUAUGAUUUUGC [2033-2051](19/19) 26 GUAAGAAGCCAGAUGUUAA UUAACAUCUGGCUUCUUAC [1903-1921](19/19) 27 UAAAGUGGCUGCUCAGAAU AUUCUGAGCAGCCACUUUA [1634-1652](19/19) 28 AGAAUAAAGUGGCUGCUCA UGAGCAGCCACUUUAUUCU [1630-1648](19/19) 29 AGGAAAGACAAGAACAACU AGUUGUUCUUGUCUUUCCU [280-298](19/19) 30 AAAGGAAAGACAAGAACAA UUGUUCUUGUCUUUCCUUU [278-296](19/19) 31 AACUUGAAAAGGAAAGACA UGUCUUUCCUUUUCAAGUU [271-289](19/19) 32 GACAAACAUUCAAGCCGCU AGCGGCUUGAAUGUUUGUC [1443-1461](19/19) 33 GACUCCGGCAUUUCACUAA UUAGUGAAAUGCCGGAGUC [1140-1158](19/19) 34 ACCAAAACCACCCUGAAAG CUUUCAGGGUGGUUUUGGU [1099-1117](19/19) 35 ACUCUCUGAACUUCUAAAU AUUUAGAAGUUCAGAGAGU [1037-1055](19/19) 36 GACAGUGAACUCAUUAAAU AUUUAAUGAGUUCACUGUC [911-929](19/19) 37 AAGUUUGGGAGGAGCUAUU AAUAGCUCCUCCCAAACUU [670-688](19/19) 38 UUGUAGAUGACAAUGAGGU ACCUCAUUGUCAUCUACAA [499-517](19/19) 39 AAACUAACCACUAUGUACU AGUACAUAGUGGUUAGUUU [2273-2291](19/19) 40 CAAAAUCAUAGCCAAAACU AGUUUUGGCUAUGAUUUUG [2034-2052](19/19) 41 UAAGAAGCCAGAUGUUAAG CUUAACAUCUGGCUUCUUA [1904-1922](19/19) 42 ACGUGAUGAAGAUGGAAAA UUUUCCAUCUUCAUCACGU [1817-1835](19/19) 43 CUGGAAAAUAUAGUAGAAC GUUCUACUAUAUUUUCCAG [1668-1686](19/19) 44 GAAAAGGAAAGACAAGAAC GUUCUUGUCUUUCCUUUUC [276-294](19/19) 45 UGAAAAGGAAAGACAAGAA UUCUUGUCUUUCCUUUUCA [275-293](19/19) 46 UGGAGACACACUACUUGGC GCCAAGUAGUGUGUCUCCA [1211-1229](19/19) 47 UCAACCAAAACCACCCUGA UCAGGGUGGUUUUGGUUGA [1096-1114](19/19) 48 CCCUCACCUGCUACUUUAA UUAAAGUAGCAGGUGAGGG [1011-1029](19/19) 49 UGACAGUGAACUCAUUAAA UUUAAUGAGUUCACUGUCA [910-928](19/19) 50 UUGGAGGCAAGAUAUAGAU AUCUAUAUCUUGCCUCCAA [182-200](19/19) 51 GAGCUAUUAUCCAUUCCUG CAGGAAUGGAUAAUAGCUC [681-699](19/19) 52 UAGAUGACAAUGAGGUUUC GAAACCUCAUUGUCAUCUA [502-520](19/19) 53 UGCCCACAUUCCCAAAUCA UGAUUUGGGAAUGUGGGCA [428-446](19/19) 54 AGCCAAAACUAGUAUAGAA UUCUAUACUAGUUUUGGCU [2043-2061](19/19) 55 AAAGUAAGAAGCCAGAUGU ACAUCUGGCUUCUUACUUU [1900-1918](19/19) 56 CCAAAAGUAAGAAGCCAGA UCUGGCUUCUUACUUUUGG [1897-1915](19/19) 57 CCCAAAAGUAAGAAGCCAG CUGGCUUCUUACUUUUGGG [1896-1914](19/19) 58 UACGUGAUGAAGAUGGAAA UUUCCAUCUUCAUCACGUA [1816-1834](19/19) 59 GCUCAAAGAAAAAGGAGAA UUCUCCUUUUUCUUUGAGC [1730-1748](19/19) 60 AACUAGAGCAAGAUUUAGA UCUAAAUCUUGCUCUAGUU [1684-1702](19/19) 61 AAGUGGCUGCUCAGAAUUG CAAUUCUGAGCAGCCACUU [1636-1654](19/19) 62 GUAGGAGGGGUAAGAAUAA UUAUUCUUACCCCUCCUAC [1618-1636](19/19) 63 UCAACUUGCAUUAAUUCGG CCGAAUUAAUGCAAGUUGA [1592-1610](19/19) 64 AAAGACAAGAACAACUCCA UGGAGUUGUUCUUGUCUUU [283-301](19/19) 65 AGAAAGAAUUGCCUGUAAG CUUACAGGCAAUUCUUUCU [1393-1411](19/19) 66 ACACACCAGAGAAAGAAUU AAUUCUUUCUCUGGUGUGU [1384-1402](19/19) 67 AACACCAGUACAUUCUUCU AGAAGAAUGUACUGGUGUU [1295-1313](19/19) 68 AAACACCAGUACAUUCUUC GAAGAAUGUACUGGUGUUU [1294-1312](19/19) 69 ACAGAAUGGUCCUAAAACA UGUUUUAGGACCAUUCUGU [1280-1298](19/19) 70 AAACCACCCUGAAAGCACA UGUGCUUUCAGGGUGGUUU [1103-1121](19/19) 71 AACCAAAACCACCCUGAAA UUUCAGGGUGGUUUUGGUU [1098-1116](19/19) 72 UCUCUGAACUUCUAAAUGG CCAUUUAGAAGUUCAGAGA [1039-1057](19/19) 73 UUCACUCUCUGAACUUCUA UAGAAGUUCAGAGAGUGAA [1034-1052](19/19) 74 GUAGUCCACAUUUUCUUAA UUAAGAAAAUGUGGACUAC [838-856](19/19) 75 UUGAAAAUGACAAGCUGGU ACCAGCUUGUCAUUUUCAA [718-736](19/19) 76 CAGUCUUCAUUGCUACUAA UUAGUAGCAAUGAAGACUG [574-592](19/19) 77 AUGACAAUGAGGUUUCUUC GAAGAAACCUCAUUGUCAU [505-523](19/19) 78 AGAUGACAAUGAGGUUUCU AGAAACCUCAUUGUCAUCU [503-521](19/19) 79 GUAGAUGACAAUGAGGUUU AAACCUCAUUGUCAUCUAC [501-519](19/19) 80 UGCCAACUACUCCCAGGUU AACCUGGGAGUAGUUGGCA [410-428](19/19) 81 AAGCCAGAUGUUAAGAAAA UUUUCUUAACAUCUGGCUU [1908-1926](19/19) 82 ACCUUAUUCUCCUAGUGAA UUCACUAGGAGAAUAAGGU [1835-1853](19/19) 83 ACCUACUGAAAAAACAACU AGUUGUUUUUUCAGUAGGU [1765-1783](19/19) 84 ACAAAAGCCUUCACCUACU AGUAGGUGAAGGCUUUUGU [1753-1771](19/19) 85 UGCUCAAAGAAAAAGGAGA UCUCCUUUUUCUUUGAGCA [1729-1747](19/19) 86 CAACUUGCAUUAAUUCGGG CCCGAAUUAAUGCAAGUUG [1593-1611](19/19) 87 GGAAAGACAAGAACAACUC GAGUUGUUCUUGUCUUUCC [281-299](19/19) 88 UUGAAAAGGAAAGACAAGA UCUUGUCUUUCCUUUUCAA [274-292](19/19) 89 CAAAAGACAAACAUUCAAG CUUGAAUGUUUGUCUUUUG [1438-1456](19/19) 90 UCACAAAAGACAAACAUUC GAAUGUUUGUCUUUUGUGA [1435-1453](19/19) 91 ACCCCAUUCACAAAAGACA UGUCUUUUGUGAAUGGGGU [1428-1446](19/19) 92 AGAAUUGCCUGUAAGUCCU AGGACUUACAGGCAAUUCU [1397-1415](19/19) 93 AGCGACGGAAAGAGUAUGA UCAUACUCUUUCCGUCGCU [235-253](19/19) 94 AAGCACAGCAGAAUUCAAU AUUGAAUUCUGCUGUGCUU [1115-1133](19/19) 95 CUCUGAACUUCUAAAUGGG CCCAUUUAGAAGUUCAGAG [1040-1058](19/19) 96 UUGACAGUGAACUCAUUAA UUAAUGAGUUCACUGUCAA [909-927](19/19) 97 UAAGUCGAGAAGUAUUUGA UCAAAUACUUCUCGACUUA [208-226](19/19) 98 UGGAGUAAGUCGAGAAGUA UACUUCUCGACUUACUCCA [203-221](19/19) 99 UACUCAUCUAUACCCUCAA UUGAGGGUAUAGAUGAGUA [798-816](19/19) 100 UCCAAGUCCAGAAGCCAAA UUUGGCUUCUGGACUUGGA [752-770](19/19) 101 CUGUUGAUUUAGACGGUAU AUACCGUCUAAAUCAACAG [634-652](19/19) 102 UUGCUACUAAUCAGGCUCA UGAGCCUGAUUAGUAGCAA [583-601](19/19) 103 GCCCAGUCUUCAUUGCUAC GUAGCAAUGAAGACUGGGC [571-589](19/19) 104 CAUUCCCGUUUGUAGAUGA UCAUCUACAAACGGGAAUG [490-508](19/19) 105 AUGCUUUGUACUUUGAUGA UCAUCAAAGUACAAAGCAU [448-466](19/19) 106 CAGAUGCUUUGUACUUUGA UCAAAGUACAAAGCAUCUG [445-463](19/19) 107 AAGAGCUGGUACUAAUAAA UUUAUUAGUACCAGCUCUU [2370-2388](19/19) 108 GGACAAAAAAUGGCAUUUU AAAAUGCCAUUUUUUGUCC [2310-2328](19/19) 109 GUGAAAUGCUCAUACUUUA UAAAGUAUGAGCAUUUCAC [2001-2019](19/19) 110 AACCUUAUUCUCCUAGUGA UCACUAGGAGAAUAAGGUU [1834-1852](19/19) 111 AAAACCUUAUUCUCCUAGU ACUAGGAGAAUAAGGUUUU [1832-1850](19/19) 112 AAACAACUCAGCACCUUAU AUAAGGUGCUGAGUUGUUU [1776-1794](19/19) 113 CACCUACUGAAAAAACAAC GUUGUUUUUUCAGUAGGUG [1764-1782](19/19) 114 UUCACCUACUGAAAAAACA UGUUUUUUCAGUAGGUGAA [1762-1780](19/19) 115 ACAAGAACAACUCCAAAAG CUUUUGGAGUUGUUCUUGU [287-305](19/19) 116 UUGCAUUAAUUCGGGAUAU AUAUCCCGAAUUAAUGCAA [1597-1615](19/19) 117 AACUUGCAUUAAUUCGGGA UCCCGAAUUAAUGCAAGUU [1594-1612](19/19) 118 CACAAAAGACAAACAUUCA UGAAUGUUUGUCUUUUGUG [1436-1454](19/19) 119 CCCAUUCACAAAAGACAAA UUUGUCUUUUGUGAAUGGG [1430-1448](19/19) 120 GAACACACCAGAGAAAGAA UUCUUUCUCUGGUGUGUUC [1382-1400](19/19) 121 ACGGUCCACAGCUCAUCAU AUGAUGAGCUGUGGACCGU [97-115](19/19) 122 UGAGAACACACCAGAGAAA UUUCUCUGGUGUGUUCUCA [1379-1397](19/19) 123 GUGAUUCUGAAGUGGAAGA UCUUCCACUUCAGAAUCAC [1234-1252](19/19) 124 ACAGCAGAAUUCAAUGAUU AAUCAUUGAAUUCUGCUGU [1119-1137](19/19) 125 GCACAGCAGAAUUCAAUGA UCAUUGAAUUCUGCUGUGC [1117-1135](19/19) 126 CUCAUUAAAUUCAGAUGCC GGCAUCUGAAUUUAAUGAG [920-938](19/19) 127 ACUCAUUAAAUUCAGAUGC GCAUCUGAAUUUAAUGAGU [919-937](19/19) 128 AACCAGUUGACAGUGAACU AGUUCACUGUCAACUGGUU [903-921](19/19) 129 UCCUUCAGCAGCAUCCUCU AGAGGAUGCUGCUGAAGGA [870-888](19/19) 130 GAGGAUUCCUUCAGCAGCA UGCUGCUGAAGGAAUCCUC [864-882](19/19) 131 AGUAAGUCGAGAAGUAUUU AAAUACUUCUCGACUUACU [206-224](19/19) 132 GAGUAAGUCGAGAAGUAUU AAUACUUCUCGACUUACUC [205-223](19/19) 133 ACUCAUCUAUACCCUCAAU AUUGAGGGUAUAGAUGAGU [799-817](19/19) 134 UUACUCAUCUAUACCCUCA UGAGGGUAUAGAUGAGUAA [797-815](19/19) 135 UGAGUUACAGUGUCUUAAU AUUAAGACACUGUAACUCA [698-716](19/19) 136 UUUGGAGGCAAGAUAUAGA UCUAUAUCUUGCCUCCAAA [181-199](19/19) 137 AGGACAUUGAGCAAGUUUG CAAACUUGCUCAAUGUCCU [658-676](19/19) 138 UAGACGGUAUGCAACAGGA UCCUGUUGCAUACCGUCUA [643-661](19/19) 139 UUGAUUUAGACGGUAUGCA UGCAUACCGUCUAAAUCAA [637-655](19/19) 140 UUCAUUGCUACUAAUCAGG CCUGAUUAGUAGCAAUGAA [579-597](19/19) 141 AGUCUUCAUUGCUACUAAU AUUAGUAGCAAUGAAGACU [575-593](19/19) 142 CGGCUACGUUUCAGUCACU AGUGACUGAAACGUAGCCG [523-541](19/19) 143 AGGACAUGGAUUUGAUUGA UCAAUCAAAUCCAUGUCCU [157-175](19/19) 144 GCCCACAUUCCCAAAUCAG CUGAUUUGGGAAUGUGGGC [429-447](19/19) 145 AGCAGGACAUGGAUUUGAU AUCAAAUCCAUGUCCUGCU [154-172](19/19) 146 AGCUGGUACUAAUAAAGGA UCCUUUAUUAGUACCAGCU [2373-2391](19/19) 147 GAGCUGGUACUAAUAAAGG CCUUUAUUAGUACCAGCUC [2372-2390](19/19) 148 AGAGCUGGUACUAAUAAAG CUUUAUUAGUACCAGCUCU [2371-2389](19/19) 149 CUAACCACUAUGUACUUUU AAAAGUACAUAGUGGUUAG [2276-2294](19/19) 150 ACAUCUGGCUAAAAAGAAA UUUCUUUUUAGCCAGAUGU [2246-2264](19/19) 151 CUGUUCUUAUGUCAUUUGU ACAAAUGACAUAAGAACAG [2199-2217](19/19) 152 UGGGCUAGUUUCUGUGUAA UUACACAGAAACUAGCCCA [2152-2170](19/19) 153 GUAAACAAUUUCUUAGGAC GUCCUAAGAAAUUGUUUAC [2127-2145](19/19) 154 CUGUAAACAAUUUCUUAGG CCUAAGAAAUUGUUUACAG [2125-2143](19/19) 155 GUCAGUAUGUUGAAUCAGU ACUGAUUCAACAUACUGAC [2093-2111](19/19) 156 GGCAAUGUUUUCCUUGUUC GAACAAGGAAAACAUUGCC [1878-1896](19/19) 157 GCACCUUAUAUCUCGAAGU ACUUCGAGAUAUAAGGUGC [1786-1804](19/19) 158 GAAAAAACAACUCAGCACC GGUGCUGAGUUGUUUUUUC [1772-1790](19/19) 159 AGAGCAAGAUUUAGAUCAU AUGAUCUAAAUCUUGCUCU [1688-1706](19/19) 160 UAGAGCAAGAUUUAGAUCA UGAUCUAAAUCUUGCUCUA [1687-1705](19/19) 161 UAGAACUAGAGCAAGAUUU AAAUCUUGCUCUAGUUCUA [1681-1699](19/19) 162 ACGUAGGAGGGGUAAGAAU AUUCUUACCCCUCCUACGU [1616-1634](19/19) 163 UCCCUGUUGUUGACUUCAA UUGAAGUCAACAACAGGGA [1540-1558](19/19) 164 UCCCUGUAGAAAAAAUCAU AUGAUUUUUUCUACAGGGA [1516-1534](19/19) 165 UCACAAGAGAUGAACUUAG CUAAGUUCAUCUCUUGUGA [1474-1492](19/19) 166 AGAGAAAGAAUUGCCUGUA UACAGGCAAUUCUUUCUCU [1391-1409](19/19) 167 ACCAGAGAAAGAAUUGCCU AGGCAAUUCUUUCUCUGGU [1388-1406](19/19) 168 CACCAGAGAAAGAAUUGCC GGCAAUUCUUUCUCUGGUG [1387-1405](19/19) 169 AGUAUGAGCUGGAAAAACA UGUUUUUCCAGCUCAUACU [247-265](19/19) 170 GUACAUUCUUCUGGGGAUA UAUCCCCAGAAGAAUGUAC [1302-1320](19/19) 171 CAAACAGAAUGGUCCUAAA UUUAGGACCAUUCUGUUUG [1277-1295](19/19) 172 CUGGAAGUGUCAAACAGAA UUCUGUUUGACACUUCCAG [1267-1285](19/19) 173 GGAAAGAGUAUGAGCUGGA UCCAGCUCAUACUCUUUCC [241-259](19/19) 174 CAGUGAUUCUGAAGUGGAA UUCCACUUCAGAAUCACUG [1232-1250](19/19) 175 GACGGAAAGAGUAUGAGCU AGCUCAUACUCUUUCCGUC [238-256](19/19) 176 CACAGCAGAAUUCAAUGAU AUCAUUGAAUUCUGCUGUG [1118-1136](19/19) 177 GCCACAGUCAACACAGAUU AAUCUGUGUUGACUGUGGC [936-954](19/19) 178 UAAAUUCAGAUGCCACAGU ACUGUGGCAUCUGAAUUUA [925-943](19/19) 179 CCAGUUGACAGUGAACUCA UGAGUUCACUGUCAACUGG [905-923](19/19) 180 GUCGAGAAGUAUUUGACUU AAGUCAAAUACUUCUCGAC [211-229](19/19) 181 CUGUAGUCCACAUUUUCUU AAGAAAAUGUGGACUACAG [836-854](19/19) 182 GGAGUAAGUCGAGAAGUAU AUACUUCUCGACUUACUCC [204-222](19/19) 183 GACAGAAGUUGACAAUUAU AUAAUUGUCAACUUCUGUC [773-791](19/19) 184 GAGACUACCAUGGUUCCAA UUGGAACCAUGGUAGUCUC [738-756](19/19) 185 CUAUUAUCCAUUCCUGAGU ACUCAGGAAUGGAUAAUAG [684-702](19/19) 186 GACGGUAUGCAACAGGACA UGUCCUGUUGCAUACCGUC [645-663](19/19) 187 CGAGAGCCCAGUCUUCAUU AAUGAAGACUGGGCUCUCG [566-584](19/19) 188 UUGAUUGACAUACUUUGGA UCCAAAGUAUGUCAAUCAA [168-186](19/19) 189 GUCACUUGUUCCUGAUAUU AAUAUCAGGAACAAGUGAC [536-554](19/19) 190 UCGGCUACGUUUCAGUCAC GUGACUGAAACGUAGCCGA [522-540](19/19) 191 GAGGUUUCUUCGGCUACGU ACGUAGCCGAAGAAACCUC [513-531](19/19) 192 GACAUUCCCGUUUGUAGAU AUCUACAAACGGGAAUGUC [488-506](19/19) 193 GACAUGGAUUUGAUUGACA UGUCAAUCAAAUCCAUGUC [159-177](19/19) 194 UCAGAUGCUUUGUACUUUG CAAAGUACAAAGCAUCUGA [444-462](19/19) 195 CAGCAGGACAUGGAUUUGA UCAAAUCCAUGUCCUGCUG [153-171](19/19) 196 AUGGACAAAAAAUGGCAUU AAUGCCAUUUUUUGUCCAU [2308-2326](19/19) 197 GUAUGGACAAAAAAUGGCA UGCCAUUUUUUGUCCAUAC [2306-2324](19/19) 198 CAUCUGGCUAAAAAGAAAU AUUUCUUUUUAGCCAGAUG [2247-2265](19/19) 199 GACAUCUGGCUAAAAAGAA UUCUUUUUAGCCAGAUGUC [2245-2263](19/19) 200 GAGCUAGUUUUUUUGUACU AGUACAAAAAAACUAGCUC [1956-1974](19/19) 201 GAAGAUGGAAAACCUUAUU AAUAAGGUUUUCCAUCUUC [1824-1842](19/19) 202 GCUACGUGAUGAAGAUGGA UCCAUCUUCAUCACGUAGC [1814-1832](19/19) 203 UGCUACGUGAUGAAGAUGG CCAUCUUCAUCACGUAGCA [1813-1831](19/19) 204 GAGCAAGAUUUAGAUCAUU AAUGAUCUAAAUCUUGCUC [1689-1707](19/19) 205 ACUAGAGCAAGAUUUAGAU AUCUAAAUCUUGCUCUAGU [1685-1703](19/19) 206 UAGUAGAACUAGAGCAAGA UCUUGCUCUAGUUCUACUA [1678-1696](19/19) 207 AUAUCCCAUUCCCUGUAGA UCUACAGGGAAUGGGAUAU [1507-1525](19/19) 208 CCAUAUCCCAUUCCCUGUA UACAGGGAAUGGGAUAUGG [1505-1523](19/19) 209 CCCCAUUCACAAAAGACAA UUGUCUUUUGUGAAUGGGG [1429-1447](19/19) 210 ACACCAGAGAAAGAAUUGC GCAAUUCUUUCUCUGGUGU [1386-1404](19/19) 211 UCAAACAGAAUGGUCCUAA UUAGGACCAUUCUGUUUGA [1276-1294](19/19) 212 AAGAGUAUGAGCUGGAAAA UUUUCCAGCUCAUACUCUU [244-262](19/19) 213 UGCCCCUGGAAGUGUCAAA UUUGACACUUCCAGGGGCA [1262-1280](19/19) 214 GCGACGGAAAGAGUAUGAG CUCAUACUCUUUCCGUCGC [236-254](19/19) 215 CUGAACUUCUAAAUGGGCC GGCCCAUUUAGAAGUUCAG [1042-1060](19/19) 216 CCACAGUCAACACAGAUUU AAAUCUGUGUUGACUGUGG [937-955](19/19) 217 AUGCUUUUGAGGAUUCCUU AAGGAAUCCUCAAAAGCAU [856-874](19/19) 218 CUCAAUGGAAAAAGAAGUA UACUUCUUUUUCCAUUGAG [812-830](19/19) 219 CUCAUCUAUACCCUCAAUG CAUUGAGGGUAUAGAUGAG [800-818](19/19) 220 ACAUUGAGCAAGUUUGGGA UCCCAAACUUGCUCAAUGU [661-679](19/19) 221 AACAGGACAUUGAGCAAGU ACUUGCUCAAUGUCCUGUU [655-673](19/19) 222 UUAGACGGUAUGCAACAGG CCUGUUGCAUACCGUCUAA [642-660](19/19) 223 ACCUGAAACUUCUGUUGCU AGCAACAGAAGUUUCAGGU [605-623](19/19) 224 CCAGUCUUCAUUGCUACUA UAGUAGCAAUGAAGACUGG [573-591](19/19) 225 AGAGCCCAGUCUUCAUUGC GCAAUGAAGACUGGGCUCU [568-586](19/19) 226 AGUCACUUGUUCCUGAUAU AUAUCAGGAACAAGUGACU [535-553](19/19) 227 UGGAUUUGAUUGACAUACU AGUAUGUCAAUCAAAUCCA [163-181](19/19) 228 CAAUGAGGUUUCUUCGGCU AGCCGAAGAAACCUCAUUG [509-527](19/19) 229 ACAAUGAGGUUUCUUCGGC GCCGAAGAAACCUCAUUGU [508-526](19/19) 230 AGACAUUCCCGUUUGUAGA UCUACAAACGGGAAUGUCU [487-505](19/19) 231 GCUUUGUACUUUGAUGACU AGUCAUCAAAGUACAAAGC [450-468](19/19) 232 CCCACAUUCCCAAAUCAGA UCUGAUUUGGGAAUGUGGG [430-448](19/19) 233 ACAACUAGAUGAAGAGACA UGUCUCUUCAUCUAGUUGU [335-353](19/19) 234 UACAACUAGAUGAAGAGAC GUCUCUUCAUCUAGUUGUA [334-352](19/19) 235 UUUAAGAGCUGGUACUAAU AUUAGUACCAGCUCUUAAA [2367-2385](19/19) 236 UGGACAAAAAAUGGCAUUU AAAUGCCAUUUUUUGUCCA [2309-2327](19/19) 237 GGGCUAGUUUCUGUGUAAG CUUACACAGAAACUAGCCC [2153-2171](19/19) 238 UUGGGCUAGUUUCUGUGUA UACACAGAAACUAGCCCAA [2151-2169](19/19) 239 GUGUCAGUAUGUUGAAUCA UGAUUCAACAUACUGACAC [2091-2109](19/19) 240 GAUGUGAAAUGCUCAUACU AGUAUGAGCAUUUCACAUC [1998-2016](19/19) 241 CUCCUACUGUGAUGUGAAA UUUCACAUCACAGUAGGAG [1988-2006](19/19) 242 AGAUUUAGGAGGAUUUGAC GUCAAAUCCUCCUAAAUCU [1930-1948](19/19) 243 GCAAUGUUUUCCUUGUUCC GGAACAAGGAAAACAUUGC [1879-1897](19/19) 244 AACAAGAGAUGGCAAUGUU AACAUUGCCAUCUCUUGUU [1868-1886](19/19) 245 GCAGCAAACAAGAGAUGGC GCCAUCUCUUGUUUGCUGC [1862-1880](19/19) 246 CUCCUAGUGAAUACUCCCU AGGGAGUAUUCACUAGGAG [1843-1861](19/19) 247 CAGCACCUUAUAUCUCGAA UUCGAGAUAUAAGGUGCUG [1784-1802](19/19) 248 UCACCUACUGAAAAAACAA UUGUUUUUUCAGUAGGUGA [1763-1781](19/19) 249 GCAAGAUUUAGAUCAUUUG CAAAUGAUCUAAAUCUUGC [1691-1709](19/19) 250 CAGAAAAAGAAAACUGGAA UUCCAGUUUUCUUUUUCUG [1655-1673](19/19) 251 AUACGUAGGAGGGGUAAGA UCUUACCCCUCCUACGUAU [1614-1632](19/19) 252 UGCAUUAAUUCGGGAUAUA UAUAUCCCGAAUUAAUGCA [1598-1616](19/19) 253 AAGCUCUCCAUAUCCCAUU AAUGGGAUAUGGAGAGCUU [1498-1516](19/19) 254 CAUUCACAAAAGACAAACA UGUUUGUCUUUUGUGAAUG [1432-1450](19/19) 255 GAGUAUGAGCUGGAAAAAC GUUUUUCCAGCUCAUACUC [246-264](19/19) 256 CAGAAUGGUCCUAAAACAC GUGUUUUAGGACCAUUCUG [1281-1299](19/19) 257 GUGUCAAACAGAAUGGUCC GGACCAUUCUGUUUGACAC [1273-1291](19/19) 258 GAAGUGUCAAACAGAAUGG CCAUUCUGUUUGACACUUC [1270-1288](19/19) 259 CUGAAGUGGAAGAGCUAGA UCUAGCUCUUCCACUUCAG [1240-1258](19/19) 260 CCAGAACACUCAGUGGAAU AUUCCACUGAGUGUUCUGG [1182-1200](19/19) 261 UUCUGACUCCGGCAUUUCA UGAAAUGCCGGAGUCAGAA [1136-1154](19/19) 262 AUGAUUCUGACUCCGGCAU AUGCCGGAGUCAGAAUCAU [1132-1150](19/19) 263 GGCCCAUUGAUGUUUCUGA UCAGAAACAUCAAUGGGCC [1057-1075](19/19) 264 CACAGAUUUUGGUGAUGAA UUCAUCACCAAAAUCUGUG [947-965](19/19) 265 CAGUUGACAGUGAACUCAU AUGAGUUCACUGUCAACUG [906-924](19/19) 266 UUAAUGCUUUUGAGGAUUC GAAUCCUCAAAAGCAUUAA [853-871](19/19) 267 UCUUAAUGCUUUUGAGGAU AUCCUCAAAAGCAUUAAGA [851-869](19/19) 268 AGUCCACAUUUUCUUAAUG CAUUAAGAAAAUGUGGACU [840-858](19/19) 269 UGACAGAAGUUGACAAUUA UAAUUGUCAACUUCUGUCA [772-790](19/19) 270 CCAAACUGACAGAAGUUGA UCAACUUCUGUCAGUUUGG [766-784](19/19) 271 GUUGAGACUACCAUGGUUC GAACCAUGGUAGUCUCAAC [735-753](19/19) 272 ACAAGCUGGUUGAGACUAC GUAGUCUCAACCAGCUUGU [727-745](19/19) 273 AUACUUUGGAGGCAAGAUA UAUCUUGCCUCCAAAGUAU [177-195](19/19) 274 GACAUACUUUGGAGGCAAG CUUGCCUCCAAAGUAUGUC [174-192](19/19) 275 GUCACCUGAAACUUCUGUU AACAGAAGUUUCAGGUGAC [602-620](19/19) 276 UCGAGAGCCCAGUCUUCAU AUGAAGACUGGGCUCUCGA [565-583](19/19) 277 GUACUUUGAUGACUGCAUG CAUGCAGUCAUCAAAGUAC [455-473](19/19) 278 UGUACUUUGAUGACUGCAU AUGCAGUCAUCAAAGUACA [454-472](19/19) 279 GGACAUGGAUUUGAUUGAC GUCAAUCAAAUCCAUGUCC [158-176](19/19) 280 GAUGCUUUGUACUUUGAUG CAUCAAAGUACAAAGCAUC [447-465](19/19) 281 AUCAGAUGCUUUGUACUUU AAAGUACAAAGCAUCUGAU [443-461](19/19) 282 UUCCCAAAUCAGAUGCUUU AAAGCAUCUGAUUUGGGAA [436-454](19/19) 283 CAUUCCCAAAUCAGAUGCU AGCAUCUGAUUUGGGAAUG [434-452](19/19) 284 CCACAUUCCCAAAUCAGAU AUCUGAUUUGGGAAUGUGG [431-449](19/19) 285 AGUUACAACUAGAUGAAGA UCUUCAUCUAGUUGUAACU [331-349](19/19) 286 UAUGGACAAAAAAUGGCAU AUGCCAUUUUUUGUCCAUA [2307-2325](19/19) 287 ACUGUAUGGACAAAAAAUG CAUUUUUUGUCCAUACAGU [2303-2321](19/19) 288 UAUGACAUCUGGCUAAAAA UUUUUAGCCAGAUGUCAUA [2242-2260](19/19) 289 ACUGUUCUUAUGUCAUUUG CAAAUGACAUAAGAACAGU [2198-2216](19/19) 290 UAGUUUCUGUGUAAGUGUA UACACUUACACAGAAACUA [2157-2175](19/19) 291 GGCUAGUUUCUGUGUAAGU ACUUACACAGAAACUAGCC [2154-2172](19/19) 292 CAAUUUCUUAGGACACCAU AUGGUGUCCUAAGAAAUUG [2132-2150](19/19) 293 GUAUGUUGAAUCAGUAGUU AACUACUGAUUCAACAUAC [2097-2115](19/19) 294 UCAGUAUGUUGAAUCAGUA UACUGAUUCAACAUACUGA [2094-2112](19/19) 295 UGAUGUGAAAUGCUCAUAC GUAUGAGCAUUUCACAUCA [1997-2015](19/19) 296 AAAGCUCCUACUGUGAUGU ACAUCACAGUAGGAGCUUU [1984-2002](19/19) 297 UACUAAAAGCUCCUACUGU ACAGUAGGAGCUUUUAGUA [1979-1997](19/19) 298 GGAUUUGACCUUUUCUGAG CUCAGAAAAGGUCAAAUCC [1940-1958](19/19) 299 AGAGAUGGCAAUGUUUUCC GGAAAACAUUGCCAUCUCU [1872-1890](19/19) 300 AAACAAGAGAUGGCAAUGU ACAUUGCCAUCUCUUGUUU [1867-1885](19/19) 301 CAGCAAACAAGAGAUGGCA UGCCAUCUCUUGUUUGCUG [1863-1881](19/19) 302 CUAGUGAAUACUCCCUGCA UGCAGGGAGUAUUCACUAG [1846-1864](19/19) 303 AAGAUGGAAAACCUUAUUC GAAUAAGGUUUUCCAUCUU [1825-1843](19/19) 304 CAGCAUGCUACGUGAUGAA UUCAUCACGUAGCAUGCUG [1808-1826](19/19) 305 CACCUUAUAUCUCGAAGUU AACUUCGAGAUAUAAGGUG [1787-1805](19/19) 306 UCAGCACCUUAUAUCUCGA UCGAGAUAUAAGGUGCUGA [1783-1801](19/19) 307 AUAUAGUAGAACUAGAGCA UGCUCUAGUUCUACUAUAU [1675-1693](19/19) 308 UUGCAGAAAAAGAAAACUG CAGUUUUCUUUUUCUGCAA [1652-1670](19/19) 309 ACGAAAUGAUGUCCAAAGA UCUUUGGACAUCAUUUCGU [1558-1576](19/19) 310 ACCUCCCUGUUGUUGACUU AAGUCAACAACAGGGAGGU [1537-1555](19/19) 311 CCCUGUAGAAAAAAUCAUU AAUGAUUUUUUCUACAGGG [1517-1535](19/19) 312 GAGAUGAACUUAGGGCAAA UUUGCCCUAAGUUCAUCUC [1480-1498](19/19) 313 CACAAGAGAUGAACUUAGG CCUAAGUUCAUCUCUUGUG [1475-1493](19/19) 314 AACCCCAUUCACAAAAGAC GUCUUUUGUGAAUGGGGUU [1427-1445](19/19) 315 AGAACACACCAGAGAAAGA UCUUUCUCUGGUGUGUUCU [1381-1399](19/19) 316 GUCAAACAGAAUGGUCCUA UAGGACCAUUCUGUUUGAC [1275-1293](19/19) 317 GGAAGUGUCAAACAGAAUG CAUUCUGUUUGACACUUCC [1269-1287](19/19) 318 UGGAAGUGUCAAACAGAAU AUUCUGUUUGACACUUCCA [1268-1286](19/19) 319 CCCUGGAAGUGUCAAACAG CUGUUUGACACUUCCAGGG [1265-1283](19/19) 320 GUGGAAGAGCUAGAUAGUG CACUAUCUAGCUCUUCCAC [1245-1263](19/19) 321 ACCAGAACACUCAGUGGAA UUCCACUGAGUGUUCUGGU [1181-1199](19/19) 322 CAGCGACGGAAAGAGUAUG CAUACUCUUUCCGUCGCUG [234-252](19/19) 323 UGAUCUAUCACUUUGCAAA UUUGCAAAGUGAUAGAUCA [1073-1091](19/19) 324 GCCCAUUGAUGUUUCUGAU AUCAGAAACAUCAAUGGGC [1058-1076](19/19) 325 AGUCAACACAGAUUUUGGU ACCAAAAUCUGUGUUGACU [941-959](19/19) 326 AGUUGACAGUGAACUCAUU AAUGAGUUCACUGUCAACU [907-925](19/19) 327 ACUGUAGUCCACAUUUUCU AGAAAAUGUGGACUACAGU [835-853](19/19) 328 CAAUGGAAAAAGAAGUAGG CCUACUUCUUUUUCCAUUG [814-832](19/19) 329 CAAACUGACAGAAGUUGAC GUCAACUUCUGUCAGUUUG [767-785](19/19) 330 AGACUACCAUGGUUCCAAG CUUGGAACCAUGGUAGUCU [739-757](19/19) 331 ACAGGACAUUGAGCAAGUU AACUUGCUCAAUGUCCUGU [656-674](19/19) 332 UGACAUACUUUGGAGGCAA UUGCCUCCAAAGUAUGUCA [173-191](19/19) 333 ACGUUUCAGUCACUUGUUC GAACAAGUGACUGAAACGU [528-546](19/19) 334 CUACGUUUCAGUCACUUGU ACAAGUGACUGAAACGUAG [526-544](19/19) 335 GGCUACGUUUCAGUCACUU AAGUGACUGAAACGUAGCC [524-542](19/19) 336 GGAUUUGAUUGACAUACUU AAGUAUGUCAAUCAAAUCC [164-182](19/19) 337 UCUUCGGCUACGUUUCAGU ACUGAAACGUAGCCGAAGA [519-537](19/19) 338 CAGUCAGAAACCAGUGGAU AUCCACUGGUUUCUGACUG [390-408](19/19) 339 UGAAGAGACAGGUGAAUUU AAAUUCACCUGUCUCUUCA [344-362](19/19) 340 UAGAUGAAGAGACAGGUGA UCACCUGUCUCUUCAUCUA [340-358](19/19) 341 UAAGAGCUGGUACUAAUAA UUAUUAGUACCAGCUCUUA [2369-2387](19/19) 342 CUGUAUGGACAAAAAAUGG CCAUUUUUUGUCCAUACAG [2304-2322](19/19) 343 UGGAGUGUCAGUAUGUUGA UCAACAUACUGACACUCCA [2087-2105](19/19) 344 UGUGAAAUGCUCAUACUUU AAAGUAUGAGCAUUUCACA [2000-2018](19/19) 345 GUGAUGUGAAAUGCUCAUA UAUGAGCAUUUCACAUCAC [1996-2014](19/19) 346 AGCUCCUACUGUGAUGUGA UCACAUCACAGUAGGAGCU [1986-2004](19/19) 347 UCCUAGUGAAUACUCCCUG CAGGGAGUAUUCACUAGGA [1844-1862](19/19) 348 UUCUCCUAGUGAAUACUCC GGAGUAUUCACUAGGAGAA [1841-1859](19/19) 349 CAUGCUACGUGAUGAAGAU AUCUUCAUCACGUAGCAUG [1811-1829](19/19) 350 GCAUGCUACGUGAUGAAGA UCUUCAUCACGUAGCAUGC [1810-1828](19/19) 351 AGCACCUUAUAUCUCGAAG CUUCGAGAUAUAAGGUGCU [1785-1803](19/19) 352 AGAAAAAGGAGAAAAUGAC GUCAUUUUCUCCUUUUUCU [1736-1754](19/19) 353 UGCAGAAAAAGAAAACUGG CCAGUUUUCUUUUUCUGCA [1653-1671](19/19) 354 UCAUUAACCUCCCUGUUGU ACAACAGGGAGGUUAAUGA [1531-1549](19/19) 355 AAGUGUCAAACAGAAUGGU ACCAUUCUGUUUGACACUU [1271-1289](19/19) 356 GAAAGAGUAUGAGCUGGAA UUCCAGCUCAUACUCUUUC [242-260](19/19) 357 AAAGCACAGCAGAAUUCAA UUGAAUUCUGCUGUGCUUU [1114-1132](19/19) 358 AUGUUUCUGAUCUAUCACU AGUGAUAGAUCAGAAACAU [1066-1084](19/19) 359 UGGGCCCAUUGAUGUUUCU AGAAACAUCAAUGGGCCCA [1055-1073](19/19) 360 CAGUCAACACAGAUUUUGG CCAAAAUCUGUGUUGACUG [940-958](19/19) 361 ACAGUCAACACAGAUUUUG CAAAAUCUGUGUUGACUGU [939-957](19/19) 362 CACAGUCAACACAGAUUUU AAAAUCUGUGUUGACUGUG [938-956](19/19) 363 ACAGAAGACCCCAACCAGU ACUGGUUGGGGUCUUCUGU [891-909](19/19) 364 AGGAUUCCUUCAGCAGCAU AUGCUGCUGAAGGAAUCCU [865-883](19/19) 365 AAUGGAAAAAGAAGUAGGU ACCUACUUCUUUUUCCAUU [815-833](19/19) 366 AUGACAAGCUGGUUGAGAC GUCUCAACCAGCUUGUCAU [724-742](19/19) 367 UUGAGCAAGUUUGGGAGGA UCCUCCCAAACUUGCUCAA [664-682](19/19) 368 CUGAAACUUCUGUUGCUCA UGAGCAACAGAAGUUUCAG [607-625](19/19) 369 UCACCUGAAACUUCUGUUG CAACAGAAGUUUCAGGUGA [603-621](19/19) 370 UUGACAUACUUUGGAGGCA UGCCUCCAAAGUAUGUCAA [172-190](19/19) 371 UGAUUGACAUACUUUGGAG CUCCAAAGUAUGUCAAUCA [169-187](19/19) 372 AGGUUUCUUCGGCUACGUU AACGUAGCCGAAGAAACCU [514-532](19/19) 373 AUGGAUUUGAUUGACAUAC GUAUGUCAAUCAAAUCCAU [162-180](19/19) 374 ACAUGGAUUUGAUUGACAU AUGUCAAUCAAAUCCAUGU [160-178](19/19) 375 UUCUCCCAAUUCAGCCAGC GCUGGCUGAAUUGGGAGAA [361-379](19/19) 376 AAGAGACAGGUGAAUUUCU AGAAAUUCACCUGUCUCUU [346-364](19/19) 377 GAUGAAGAGACAGGUGAAU AUUCACCUGUCUCUUCAUC [342-360](19/19) 378 AACUAGAUGAAGAGACAGG CCUGUCUCUUCAUCUAGUU [337-355](19/19) 379 UUGCAAAACUAACCACUAU AUAGUGGUUAGUUUUGCAA [2268-2286](19/19) 380 UGACAUCUGGCUAAAAAGA UCUUUUUAGCCAGAUGUCA [2244-2262](19/19) 381 GAGUGUCAGUAUGUUGAAU AUUCAACAUACUGACACUC [2089-2107](19/19) 382 AAGCAUUGGAGUGUCAGUA UACUGACACUCCAAUGCUU [2081-2099](19/19) 383 GCUCCUACUGUGAUGUGAA UUCACAUCACAGUAGGAGC [1987-2005](19/19) 384 GACCUUUUCUGAGCUAGUU AACUAGCUCAGAAAAGGUC [1946-1964](19/19) 385 UGACCUUUUCUGAGCUAGU ACUAGCUCAGAAAAGGUCA [1945-1963](19/19) 386 UCAUGAUGGACUUGGAGCU AGCUCCAAGUCCAUCAUGA [112-130](19/19) 387 CCUUAUAUCUCGAAGUUUU AAAACUUCGAGAUAUAAGG [1789-1807](19/19) 388 CUCAGCACCUUAUAUCUCG CGAGAUAUAAGGUGCUGAG [1782-1800](19/19) 389 GACAAGAACAACUCCAAAA UUUUGGAGUUGUUCUUGUC [286-304](19/19) 390 UCGGGAUAUACGUAGGAGG CCUCCUACGUAUAUCCCGA [1607-1625](19/19) 391 UAAUUCGGGAUAUACGUAG CUACGUAUAUCCCGAAUUA [1603-1621](19/19) 392 GAAAUGAUGUCCAAAGAGC GCUCUUUGGACAUCAUUUC [1560-1578](19/19) 393 AACGAAAUGAUGUCCAAAG CUUUGGACAUCAUUUCGUU [1557-1575](19/19) 394 GAAAAAAUCAUUAACCUCC GGAGGUUAAUGAUUUUUUC [1524-1542](19/19) 395 AGCUCUCCAUAUCCCAUUC GAAUGGGAUAUGGAGAGCU [1499-1517](19/19) 396 GCAAAAGCUCUCCAUAUCC GGAUAUGGAGAGCUUUUGC [1494-1512](19/19) 397 AAGAGAUGAACUUAGGGCA UGCCCUAAGUUCAUCUCUU [1478-1496](19/19) 398 CUGGAAAAACAGAAAAAAC GUUUUUUCUGUUUUUCCAG [255-273](19/19) 399 GAGCUGGAAAAACAGAAAA UUUUCUGUUUUUCCAGCUC [252-270](19/19) 400 CCCCUGGAAGUGUCAAACA UGUUUGACACUUCCAGGGG [1264-1282](19/19) 401 AUUCUGAAGUGGAAGAGCU AGCUCUUCCACUUCAGAAU [1237-1255](19/19) 402 CAUCACCAGAACACUCAGU ACUGAGUGUUCUGGUGAUG [1177-1195](19/19) 403 CUGAUCUAUCACUUUGCAA UUGCAAAGUGAUAGAUCAG [1072-1090](19/19) 404 UCUGAUCUAUCACUUUGCA UGCAAAGUGAUAGAUCAGA [1071-1089](19/19) 405 UUCUGAUCUAUCACUUUGC GCAAAGUGAUAGAUCAGAA [1070-1088](19/19) 406 GAGCCCAGUAUCAGCAACA UGUUGCUGAUACUGGGCUC [987-1005](19/19) 407 GAAGUAGGUAACUGUAGUC GACUACAGUUACCUACUUC [825-843](19/19) 408 AGAAGUAGGUAACUGUAGU ACUACAGUUACCUACUUCU [824-842](19/19) 409 AAAAGAAGUAGGUAACUGU ACAGUUACCUACUUCUUUU [821-839](19/19) 410 GCAACAGGACAUUGAGCAA UUGCUCAAUGUCCUGUUGC [653-671](19/19) 411 CACUUGUUCCUGAUAUUCC GGAAUAUCAGGAACAAGUG [538-556](19/19) 412 GCAGGACAUGGAUUUGAUU AAUCAAAUCCAUGUCCUGC [155-173](19/19) 413 GUUUCUGUGUAAGUGUAAA UUUACACUUACACAGAAAC [2159-2177](19/19) 414 GCAUUGGAGUGUCAGUAUG CAUACUGACACUCCAAUGC [2083-2101](19/19) 415 UGUGAUGUGAAAUGCUCAU AUGAGCAUUUCACAUCACA [1995-2013](19/19) 416 ACUAGAUUUAGGAGGAUUU AAAUCCUCCUAAAUCUAGU [1927-1945](19/19) 417 CUCGAAGUUUUCAGCAUGC GCAUGCUGAAAACUUCGAG [1797-1815](19/19) 418 AUCUCGAAGUUUUCAGCAU AUGCUGAAAACUUCGAGAU [1795-1813](19/19) 419 GCAGAAAAAGAAAACUGGA UCCAGUUUUCUUUUUCUGC [1654-1672](19/19) 420 GGAUAUACGUAGGAGGGGU ACCCCUCCUACGUAUAUCC [1610-1628](19/19) 421 AAGACAAGAACAACUCCAA UUGGAGUUGUUCUUGUCUU [284-302](19/19) 422 AAAGAGCAGUUCAAUGAAG CUUCAUUGAACUGCUCUUU [1572-1590](19/19) 423 UCAACGAAAUGAUGUCCAA UUGGACAUCAUUUCGUUGA [1555-1573](19/19) 424 UAACCUCCCUGUUGUUGAC GUCAACAACAGGGAGGUUA [1535-1553](19/19) 425 CAAAAGCUCUCCAUAUCCC GGGAUAUGGAGAGCUUUUG [1495-1513](19/19) 426 AGAUGAACUUAGGGCAAAA UUUUGCCCUAAGUUCAUCU [1481-1499](19/19) 427 AGAGAUGAACUUAGGGCAA UUGCCCUAAGUUCAUCUCU [1479-1497](19/19) 428 AGCUGGAAAAACAGAAAAA UUUUUCUGUUUUUCCAGCU [253-271](19/19) 429 UCCUAAAACACCAGUACAU AUGUACUGGUGUUUUAGGA [1289-1307](19/19) 430 UGAUGUUUCUGAUCUAUCA UGAUAGAUCAGAAACAUCA [1064-1082](19/19) 431 CCAUUGAUGUUUCUGAUCU AGAUCAGAAACAUCAAUGG [1060-1078](19/19) 432 AGUAUGUUGAAUCAGUAGU ACUACUGAUUCAACAUACU [2096-2114](19/19) 433 UUCUGAGCUAGUUUUUUUG CAAAAAAACUAGCUCAGAA [1952-1970](19/19) 434 UCGAAGUUUUCAGCAUGCU AGCAUGCUGAAAACUUCGA [1798-1816](19/19) 435 AUUCGGGAUAUACGUAGGA UCCUACGUAUAUCCCGAAU [1605-1623](19/19) 436 UGUCCAAAGAGCAGUUCAA UUGAACUGCUCUUUGGACA [1567-1585](19/19) 437 UUCUGGGGAUAUGGUACAA UUGUACCAUAUCCCCAGAA [1310-1328](19/19) 438 CUUCUGGGGAUAUGGUACA UGUACCAUAUCCCCAGAAG [1309-1327](19/19) 439 UGUUUCUGAUCUAUCACUU AAGUGAUAGAUCAGAAACA [1067-1085](19/19) 440 ACCUGCUACUUUAAGCCAU AUGGCUUAAAGUAGCAGGU [1016-1034](19/19) 441 GUUUCAGUCACUUGUUCCU AGGAACAAGUGACUGAAAC [530-548](19/19) 442 CACAUCCAGUCAGAAACCA UGGUUUCUGACUGGAUGUG [384-402](19/19) 443 CUGGUACUAAUAAAGGAUU AAUCCUUUAUUAGUACCAG [2375-2393](19/19) 444 AGUGUCAGUAUGUUGAAUC GAUUCAACAUACUGACACU [2090-2108](19/19) 445 AAACUAGAUUUAGGAGGAU AUCCUCCUAAAUCUAGUUU [1925-1943](19/19) 446 AAAACUAGAUUUAGGAGGA UCCUCCUAAAUCUAGUUUU [1924-1942](19/19) 447 AGCUCAACUUGCAUUAAUU AAUUAAUGCAAGUUGAGCU [1589-1607](19/19) 448 AAGAGCAGUUCAAUGAAGC GCUUCAUUGAACUGCUCUU [1573-1591](19/19) 449 UGAUGUCCAAAGAGCAGUU AACUGCUCUUUGGACAUCA [1564-1582](19/19) 450 GAAAACCCCAUUCACAAAA UUUUGUGAAUGGGGUUUUC [1424-1442](19/19) 451 UGAGCUGGAAAAACAGAAA UUUCUGUUUUUCCAGCUCA [251-269](19/19) 452 UUGAUGUUUCUGAUCUAUC GAUAGAUCAGAAACAUCAA [1063-1081](19/19) 453 UUGCCCACAUUCCCAAAUC GAUUUGGGAAUGUGGGCAA [427-445](19/19) 454 GCACAUCCAGUCAGAAACC GGUUUCUGACUGGAUGUGC [383-401](19/19) 455 GCUGGUACUAAUAAAGGAU AUCCUUUAUUAGUACCAGC [2374-2392](19/19) 456 GGAGUGUCAGUAUGUUGAA UUCAACAUACUGACACUCC [2088-2106](19/19) 457 CCUUUUCUGAGCUAGUUUU AAAACUAGCUCAGAAAAGG [1948-1966](19/19) 458 CUCAAAGAAAAAGGAGAAA UUUCUCCUUUUUCUUUGAG [1731-1749](19/19) 459 AGACAAGAACAACUCCAAA UUUGGAGUUGUUCUUGUCU [285-303](19/19) 460 AUGAUGUCCAAAGAGCAGU ACUGCUCUUUGGACAUCAU [1563-1581](19/19) 461 AUGAGCUGGAAAAACAGAA UUCUGUUUUUCCAGCUCAU [250-268](19/19) 462 GUCCUAAAACACCAGUACA UGUACUGGUGUUUUAGGAC [1288-1306](19/19) 463 AUCAGCAACAGCAUGCCCU AGGGCAUGCUGUUGCUGAU [996-1014](19/19) 464 AAGCCAAACUGACAGAAGU ACUUCUGUCAGUUUGGCUU [763-781](19/19) 465 CGUUUGUAGAUGACAAUGA UCAUUGUCAUCUACAAACG [496-514](19/19) 466 GUUGCCCACAUUCCCAAAU AUUUGGGAAUGUGGGCAAC [426-444](19/19) 467 GCAAACAAGAGAUGGCAAU AUUGCCAUCUCUUGUUUGC [1865-1883](19/19) 468 AUUGCUCAAAGAAAAAGGA UCCUUUUUCUUUGAGCAAU [1727-1745](19/19) 469 AAACCCCAUUCACAAAAGA UCUUUUGUGAAUGGGGUUU [1426-1444](19/19) 470 AAGUGGAAGAGCUAGAUAG CUAUCUAGCUCUUCCACUU [1243-1261](19/19) 471 UCACUAAACACAAGUCCCA UGGGACUUGUGUUUAGUGA [1152-1170](19/19) 472 CGGCAUUUCACUAAACACA UGUGUUUAGUGAAAUGCCG [1145-1163](19/19) 473 UUUAAGCCAUUCACUCUCU AGAGAGUGAAUGGCUUAAA [1025-1043](19/19) 474 CACCUGCUACUUUAAGCCA UGGCUUAAAGUAGCAGGUG [1015-1033](19/19) 475 GGAGGAGCUAUUAUCCAUU AAUGGAUAAUAGCUCCUCC [677-695](19/19) 476 GGGAGGAGCUAUUAUCCAU AUGGAUAAUAGCUCCUCCC [676-694](19/19) 477 UCGCUCAGUUACAACUAGA UCUAGUUGUAACUGAGCGA [325-343](19/19) 478 UGAAGAUGGAAAACCUUAU AUAAGGUUUUCCAUCUUCA [1823-1841](19/19) 479 AAGCUCAACUUGCAUUAAU AUUAAUGCAAGUUGAGCUU [1588-1606](19/19) 480 AAAACCCCAUUCACAAAAG CUUUUGUGAAUGGGGUUUU [1425-1443](19/19) 481 GCUAUGGAGACACACUACU AGUAGUGUGUCUCCAUAGC [1207-1225](19/19) 482 GUAUUUGACUUCAGUCAGC GCUGACUGAAGUCAAAUAC [219-237](19/19) 483 CCUGCUACUUUAAGCCAUU AAUGGCUUAAAGUAGCAGG [1017-1035](19/19) 484 UGAGCCCAGUAUCAGCAAC GUUGCUGAUACUGGGCUCA [986-1004](19/19) 485 GAGAAGUAUUUGACUUCAG CUGAAGUCAAAUACUUCUC [214-232](19/19) 486 UGGAAAAAGAAGUAGGUAA UUACCUACUUCUUUUUCCA [817-835](19/19) 487 GAAGCCAAACUGACAGAAG CUUCUGUCAGUUUGGCUUC [762-780](19/19) 488 CAGAAGCCAAACUGACAGA UCUGUCAGUUUGGCUUCUG [760-778](19/19) 489 GAAAAUGACAAGCUGGUUG CAACCAGCUUGUCAUUUUC [720-738](19/19) 490 AAAAAGCAUUGGAGUGUCA UGACACUCCAAUGCUUUUU [2078-2096](19/19) 491 UAGGAGGAUUUGACCUUUU AAAAGGUCAAAUCCUCCUA [1935-1953](19/19) 492 AGAUGGAAAACCUUAUUCU AGAAUAAGGUUUUCCAUCU [1826-1844](19/19) 493 GAAGCUCAACUUGCAUUAA UUAAUGCAAGUUGAGCUUC [1587-1605](1919) 494 GCUCUCCAUAUCCCAUUCC GGAAUGGGAUAUGGAGAGC [1500-1518](19/19) 495 GGAAAACCCCAUUCACAAA UUUGUGAAUGGGGUUUUCC [1423-1441](19/19) 496 AGAGCACUCACGUGCAUGA UCAUGCACGUGAGUGCUCU [1351-1369](19/19) 497 ACCCUUGUCACCAUCUCAG CUGAGAUGGUGACAAGGGU [1328-1346](19/19) 498 CUACUUGGCCUCAGUGAUU AAUCACUGAGGCCAAGUAG [1221-1239](19/19) 499 GUGAACUCAUUAAAUUCAG CUGAAUUUAAUGAGUUCAC [915-933](19/19) 500 GGAAAAAGAAGUAGGUAAC GUUACCUACUUCUUUUUCC [818-836](19/19) 501 CCCUGUCGAAAAAAUCAUU AAUGAUUUUUUCGACAGGG 502 UCCCUGUCGAAAAAAUCAU AUGAUUUUUUCGACAGGGA 503 CCUGUCGAAAAAAUCAUUA UAAUGAUUUUUUCGACAGG

TABLE B 21-mers siRNAs specific to Nrf2 gene (SEQ ID NOS: 1007-2006) Sense siRNA Antisense siRNA Human 20149575 1 UAGGAGGGGUAAGAAUAAAGU ACUUUAUUCUUACCCCUCCUA [1619-1639](21/21) 2 AGGGGUAAGAAUAAAGUGGCU AGCCACUUUAUUCUUACCCCU [1623-1643](21/21) 3 AAGAAGCCAGAUGUUAAGAAA UUUCUUAACAUCUGGCUUCUU [1905-1925](21/21) 4 AAAGGAGAAAAUGACAAAAGC GCUUUUGUCAUUUUCUCCUUU [1740-1760](21/21) 5 GAGGGGUAAGAAUAAAGUGGC GCCACUUUAUUCUUACCCCUC [1622-1642](21/21) 6 GUAGGAGGGGUAAGAAUAAAG CUUUAUUCUUACCCCUCCUAC [1618-1638](21/21) 7 AUAAAGUGGCUGCUCAGAAUU AAUUCUGAGCAGCCACUUUAU [1633-1653](21/21) 8 AGGAGGGGUAAGAAUAAAGUG CACUUUAUUCUUACCCCUCCU [1620-1640](21/21) 9 GAGAAAGAAUUGCCUGUAAGU ACUUACAGGCAAUUCUUUCUC [1392-1412](21/21) 10 UCACUCUCUGAACUUCUAAAU AUUUAGAAGUUCAGAGAGUGA [1035-1055](21/21) 11 GACAUUCCCGUUUGUAGAUGA UCAUCUACAAACGGGAAUGUC [488-508](21/21) 12 AGAAGCCAGAUGUUAAGAAAA UUUUCUUAACAUCUGGCUUCU [1906-1926](21/21) 13 CUCAGAAUUGCAGAAAAAGAA UUCUUUUUCUGCAAUUCUGAG [1645-1665](21/21) 14 AAGAAUAAAGUGGCUGCUCAG CUGAGCAGCCACUUUAUUCUU [1629-1649](21/21) 15 GGGUAAGAAUAAAGUGGCUGC GCAGCCACUUUAUUCUUACCC [1625-1645](21/21) 16 UGAAAAGGAAAGACAAGAACA UGUUCUUGUCUUUCCUUUUCA [275-295](21/21) 17 GCCCUCACCUGCUACUUUAAG CUUAAAGUAGCAGGUGAGGGC [1010-1030](21/21) 18 GUAGAUGACAAUGAGGUUUCU AGAAACCUCAUUGUCAUCUAC [501-521](21/21) 19 GUAAGAAGCCAGAUGUUAAGA UCUUAACAUCUGGCUUCUUAC [1903-1923](21/21) 20 AAGUAAGAAGCCAGAUGUUAA UUAACAUCUGGCUUCUUACUU [1901-1921](21/21) 21 GGGGUAAGAAUAAAGUGGCUG CAGCCACUUUAUUCUUACCCC [1624-1644](21/21) 22 AGAGAAAGAAUUGCCUGUAAG CUUACAGGCAAUUCUUUCUCU [1391-1411](21/21) 23 CUCUCUGAACUUCUAAAUGGG CCCAUUUAGAAGUUCAGAGAG [1038-1058](21/21) 24 UUACUCAUCUAUACCCUCAAU AUUGAGGGUAUAGAUGAGUAA [797-817](21/21) 25 CCAGUCUUCAUUGCUACUAAU AUUAGUAGCAAUGAAGACUGG [573-593](21/21) 26 UAGAUGACAAUGAGGUUUCUU AAGAAACCUCAUUGUCAUCUA [502-522](21/21) 27 ACAUUCCCGUUUGUAGAUGAC GUCAUCUACAAACGGGAAUGU [489-509](21/21) 28 GCAAAACUAACCACUAUGUAC GUACAUAGUGGUUAGUUUUGC [2270-2290](21/21) 29 AAAAGUAAGAAGCCAGAUGUU AACAUCUGGCUUCUUACUUUU [1899-1919](21/21) 30 CCCAAAAGUAAGAAGCCAGAU AUCUGGCUUCUUACUUUUGGG [1896-1916](21/21) 31 UUGCUCAAAGAAAAAGGAGAA UUCUCCUUUUUCUUUGAGCAA [1728-1748](21/21) 32 GAACUAGAGCAAGAUUUAGAU AUCUAAAUCUUGCUCUAGUUC [1683-1703](21/21) 33 GCUCAGAAUUGCAGAAAAAGA UCUUUUUCUGCAAUUCUGAGC [1644-1664](21/21) 34 UUGAAAAGGAAAGACAAGAAC GUUCUUGUCUUUCCUUUUCAA [274-294](21/21) 35 AGAACACACCAGAGAAAGAAU AUUCUUUCUCUGGUGUGUUCU [1381-1401](21/21) 36 AAAACACCAGUACAUUCUUCU AGAAGAAUGUACUGGUGUUUU [1293-1313](21/21) 37 GUCAAACAGAAUGGUCCUAAA UUUAGGACCAUUCUGUUUGAC [1275-1295](21/21) 38 AACCAAAACCACCCUGAAAGC GCUUUCAGGGUGGUUUUGGUU [1098-1118](21/21) 39 UUCAACCAAAACCACCCUGAA UUCAGGGUGGUUUUGGUUGAA [1095-1115](21/21) 40 CACUCUCUGAACUUCUAAAUG CAUUUAGAAGUUCAGAGAGUG [1036-1056](21/21) 41 GAGUAAGUCGAGAAGUAUUUG CAAAUACUUCUCGACUUACUC [205-225](21/21) 42 GACAAUGAGGUUUCUUCGGCU AGCCGAAGAAACCUCAUUGUC [507-527](21/21) 43 UCAGAUGCUUUGUACUUUGAU AUCAAAGUACAAAGCAUCUGA [444-464](21/21) 44 GACAUCUGGCUAAAAAGAAAU AUUUCUUUUUAGCCAGAUGUC [2245-2265](21/21) 45 GAAGCCAGAUGUUAAGAAAAA UUUUUCUUAACAUCUGGCUUC [1907-1927](21/21) 46 CAAAAGUAAGAAGCCAGAUGU ACAUCUGGCUUCUUACUUUUG [1898-1918](21/21) 47 AAAACCUUAUUCUCCUAGUGA UCACUAGGAGAAUAAGGUUUU [1832-1852](21/21) 48 ACGUAGGAGGGGUAAGAAUAA UUAUUCUUACCCCUCCUACGU [1616-1636](21/21) 49 AAGGAAAGACAAGAACAACUC GAGUUGUUCUUGUCUUUCCUU [279-299](21/21) 50 CACAAAAGACAAACAUUCAAG CUUGAAUGUUUGUCUUUUGUG [1436-1456](21/21) 51 CCCAUUCACAAAAGACAAACA UGUUUGUCUUUUGUGAAUGGG [1430-1450](21/21) 52 AACCCCAUUCACAAAAGACAA UUGUCUUUUGUGAAUGGGGUU [1427-1447](21/21) 53 ACACCAGAGAAAGAAUUGCCU AGGCAAUUCUUUCUCUGGUGU [1386-1406](21/21) 54 AACACACCAGAGAAAGAAUUG CAAUUCUUUCUCUGGUGUGUU [1383-1403](21/21) 55 UCAAACAGAAUGGUCCUAAAA UUUUAGGACCAUUCUGUUUGA [1276-1296](21/21) 56 UCAGCGACGGAAAGAGUAUGA UCAUACUCUUUCCGUCGCUGA [233-253](21/21) 57 GCACAGCAGAAUUCAAUGAUU AAUCAUUGAAUUCUGCUGUGC [1117-1137](21/21) 58 GCUUUCAACCAAAACCACCCU AGGGUGGUUUUGGUUGAAAGC [1092-1112](21/21) 59 GCCACAGUCAACACAGAUUUU AAAAUCUGUGUUGACUGUGGC [936-956](21/21) 60 AACUCAUUAAAUUCAGAUGCC GGCAUCUGAAUUUAAUGAGUU [918-938](21/21) 61 GUUGACAGUGAACUCAUUAAA UUUAAUGAGUUCACUGUCAAC [908-928](21/21) 62 GCUAUUAUCCAUUCCUGAGUU AACUCAGGAAUGGAUAAUAGC [683-703](21/21) 63 AAGUUUGGGAGGAGCUAUUAU AUAAUAGCUCCUCCCAAACUU [670-690](21/21) 64 CAAGUUUGGGAGGAGCUAUUA UAAUAGCUCCUCCCAAACUUG [669-689](21/21) 65 GUCUUCAUUGCUACUAAUCAG CUGAUUAGUAGCAAUGAAGAC [576-596](21/21) 66 GAUGACAAUGAGGUUUCUUCG CGAAGAAACCUCAUUGUCAUC [504-524](21/21) 67 AUCAGAUGCUUUGUACUUUGA UCAAAGUACAAAGCAUCUGAU [443-463](21/21) 68 GCCCACAUUCCCAAAUCAGAU AUCUGAUUUGGGAAUGUGGGC [429-449](21/21) 69 GUGAUGUGAAAUGCUCAUACU AGUAUGAGCAUUUCACAUCAC [1996-2016](21/21) 70 AAGCUCCUACUGUGAUGUGAA UUCACAUCACAGUAGGAGCUU [1985-2005](21/21) 71 CUACGUGAUGAAGAUGGAAAA UUUUCCAUCUUCAUCACGUAG [1815-1835](21/21) 72 AUGCUACGUGAUGAAGAUGGA UCCAUCUUCAUCACGUAGCAU [1812-1832](21/21) 73 UGAAAAAACAACUCAGCACCU AGGUGCUGAGUUGUUUUUUCA [1771-1791](21/21) 74 UCACCUACUGAAAAAACAACU AGUUGUUUUUUCAGUAGGUGA [1763-1783](21/21) 75 GUAGAACUAGAGCAAGAUUUA UAAAUCUUGCUCUAGUUCUAC [1680-1700](21/21) 76 UCAACUUGCAUUAAUUCGGGA UCCCGAAUUAAUGCAAGUUGA [1592-1612](21/21) 77 AAAGGAAAGACAAGAACAACU AGUUGUUCUUGUCUUUCCUUU [278-298](21/21) 78 CCAUUCACAAAAGACAAACAU AUGUUUGUCUUUUGUGAAUGG [1431-1451](21/21) 79 CACACCAGAGAAAGAAUUGCC GGCAAUUCUUUCUCUGGUGUG [1385-1405](21/21) 80 AACAGAAUGGUCCUAAAACAC GUGUUUUAGGACCAUUCUGUU [1279-1299](21/21) 81 GGAAAGAGUAUGAGCUGGAAA UUUCCAGCUCAUACUCUUUCC [241-261](21/21) 82 CAGUUGACAGUGAACUCAUUA UAAUGAGUUCACUGUCAACUG [906-926](21/21) 83 GGAGUAAGUCGAGAAGUAUUU AAAUACUUCUCGACUUACUCC [204-224](21/21) 84 UGGAGUAAGUCGAGAAGUAUU AAUACUUCUCGACUUACUCCA [203-223](21/21) 85 UUGGAGUAAGUCGAGAAGUAU AUACUUCUCGACUUACUCCAA [202-222](21/21) 86 CUAUUAUCCAUUCCUGAGUUA UAACUCAGGAAUGGAUAAUAG [684-704](21/21) 87 GCAACAGGACAUUGAGCAAGU ACUUGCUCAAUGUCCUGUUGC [653-673](21/21) 88 GUUGAUUUAGACGGUAUGCAA UUGCAUACCGUCUAAAUCAAC [636-656](21/21) 89 CAGUCUUCAUUGCUACUAAUC GAUUAGUAGCAAUGAAGACUG [574-594](21/21) 90 UCACAUCGAGAGCCCAGUCUU AAGACUGGGCUCUCGAUGUGA [560-580](21/21) 91 AGGACAUGGAUUUGAUUGACA UGUCAAUCAAAUCCAUGUCCU [157-177](21/21) 92 UGCCCACAUUCCCAAAUCAGA UCUGAUUUGGGAAUGUGGGCA [428-448](21/21) 93 UACUCCCAGGUUGCCCACAUU AAUGUGGGCAACCUGGGAGUA [417-437](21/21) 94 AAGAGCUGGUACUAAUAAAGG CCUUUAUUAGUACCAGCUCUU [2370-2390](21/21) 95 UAAGAGCUGGUACUAAUAAAG CUUUAUUAGUACCAGCUCUUA [2369-2389](21/21) 96 CUGUAUGGACAAAAAAUGGCA UGCCAUUUUUUGUCCAUACAG [2304-2324](21/21) 97 GGAUUUGACCUUUUCUGAGCU AGCUCAGAAAAGGUCAAAUCC [1940-1960](21/21) 98 AAACCUUAUUCUCCUAGUGAA UUCACUAGGAGAAUAAGGUUU [1833-1853](21/21) 99 AAAAAACAACUCAGCACCUUA UAAGGUGCUGAGUUGUUUUUU [1773-1793](21/21) 100 CUAGAGCAAGAUUUAGAUCAU AUGAUCUAAAUCUUGCUCUAG [1686-1706](21/21) 101 GCAGAAAAAGAAAACUGGAAA UUUCCAGUUUUCUUUUUCUGC [1654-1674](21/21) 102 AACUUGCAUUAAUUCGGGAUA UAUCCCGAAUUAAUGCAAGUU [1594-1614](21/21) 103 CAACUUGCAUUAAUUCGGGAU AUCCCGAAUUAAUGCAAGUUG [1593-1613](21/21) 104 CCAUAUCCCAUUCCCUGUAGA UCUACAGGGAAUGGGAUAUGG [1505-1525](21/21) 105 ACAAAAGACAAACAUUCAAGC GCUUGAAUGUUUGUCUUUUGU [1437-1457](21/21) 106 ACCCCAUUCACAAAAGACAAA UUUGUCUUUUGUGAAUGGGGU [1428-1448](21/21) 107 AAGAAUUGCCUGUAAGUCCUG CAGGACUUACAGGCAAUUCUU [1396-1416](21/21) 108 AAAGAAUUGCCUGUAAGUCCU AGGACUUACAGGCAAUUCUUU [1395-1415](21/21) 109 GAACACACCAGAGAAAGAAUU AAUUCUUUCUCUGGUGUGUUC [1382-1402](21/21) 110 AGAGUAUGAGCUGGAAAAACA UGUUUUUCCAGCUCAUACUCU [245-265](21/21) 111 GUAGUCCACAUUUUCUUAAUG CAUUAAGAAAAUGUGGACUAC [838-858](21/21) 112 UACUCAUCUAUACCCUCAAUG CAUUGAGGGUAUAGAUGAGUA [798-818](21/21) 113 CAGGACAUUGAGCAAGUUUGG CCAAACUUGCUCAAUGUCCUG [657-677](21/21) 114 UUCGGCUACGUUUCAGUCACU AGUGACUGAAACGUAGCCGAA [521-541](21/21) 115 ACAAUGAGGUUUCUUCGGCUA UAGCCGAAGAAACCUCAUUGU [508-528](21/21) 116 GGACAUGGAUUUGAUUGACAU AUGUCAAUCAAAUCCAUGUCC [158-178](21/21) 117 UACUGUAUGGACAAAAAAUGG CCAUUUUUUGUCCAUACAGUA [2302-2322](21/21) 118 AACAAUUUCUUAGGACACCAU AUGGUGUCCUAAGAAAUUGUU [2130-2150](21/21) 119 CUGUAAACAAUUUCUUAGGAC GUCCUAAGAAAUUGUUUACAG [2125-2145](21/21) 120 GAAGAUGGAAAACCUUAUUCU AGAAUAAGGUUUUCCAUCUUC [1824-1844](21/21) 121 UACGUGAUGAAGAUGGAAAAC GUUUUCCAUCUUCAUCACGUA [1816-1836](21/21) 122 AGCACCUUAUAUCUCGAAGUU AACUUCGAGAUAUAAGGUGCU [1785-1805](21/21) 123 AAAACAACUCAGCACCUUAUA UAUAAGGUGCUGAGUUGUUUU [1775-1795](21/21) 124 GAGCAAGAUUUAGAUCAUUUG CAAAUGAUCUAAAUCUUGCUC [1689-1709](21/21) 125 AGAACUAGAGCAAGAUUUAGA UCUAAAUCUUGCUCUAGUUCU [1682-1702](21/21) 126 UUGCAGAAAAAGAAAACUGGA UCCAGUUUUCUUUUUCUGCAA [1652-1672](21/21) 127 UCCAUAUCCCAUUCCCUGUAG CUACAGGGAAUGGGAUAUGGA [1504-1524](21/21) 128 CCCCAUUCACAAAAGACAAAC GUUUGUCUUUUGUGAAUGGGG [1429-1449](21/21) 129 GAGAACACACCAGAGAAAGAA UUCUUUCUCUGGUGUGUUCUC [1380-1400](21/21) 130 CAGUACAUUCUUCUGGGGAUA UAUCCCCAGAAGAAUGUACUG [1300-1320](21/21) 131 ACGGAAAGAGUAUGAGCUGGA UCCAGCUCAUACUCUUUCCGU [239-259](21/21) 132 CUGACUCCGGCAUUUCACUAA UUAGUGAAAUGCCGGAGUCAG [1138-1158](21/21) 133 AGCACAGCAGAAUUCAAUGAU AUCAUUGAAUUCUGCUGUGCU [1116-1136](21/21) 134 UAAAUUCAGAUGCCACAGUCA UGACUGUGGCAUCUGAAUUUA [925-945](21/21) 135 CUCAUUAAAUUCAGAUGCCAC GUGGCAUCUGAAUUUAAUGAG [920-940](21/21) 136 ACUGUAGUCCACAUUUUCUUA UAAGAAAAUGUGGACUACAGU [835-855](21/21) 137 UCAAUGGAAAAAGAAGUAGGU ACCUACUUCUUUUUCCAUUGA [813-833](21/21) 138 GAGCUAUUAUCCAUUCCUGAG CUCAGGAAUGGAUAAUAGCUC [681-701](21/21) 139 AACAGGACAUUGAGCAAGUUU AAACUUGCUCAAUGUCCUGUU [655-675](21/21) 140 UUAGACGGUAUGCAACAGGAC GUCCUGUUGCAUACCGUCUAA [642-662](21/21) 141 UUUAGACGGUAUGCAACAGGA UCCUGUUGCAUACCGUCUAAA [641-661](21/21) 142 UGAGGUUUCUUCGGCUACGUU AACGUAGCCGAAGAAACCUCA [512-532](21/21) 143 AUGAGGUUUCUUCGGCUACGU ACGUAGCCGAAGAAACCUCAU [511-531](21/21) 144 UGCUUUGUACUUUGAUGACUG CAGUCAUCAAAGUACAAAGCA [449-469](21/21) 145 AGAUGCUUUGUACUUUGAUGA UCAUCAAAGUACAAAGCAUCU [446-466](21/21) 146 CAGCAGGACAUGGAUUUGAUU AAUCAAAUCCAUGUCCUGCUG [153-173](21/21) 147 UUACAACUAGAUGAAGAGACA UGUCUCUUCAUCUAGUUGUAA [333-353](21/21) 148 AGAGCUGGUACUAAUAAAGGA UCCUUUAUUAGUACCAGCUCU [2371-2391](21/21) 149 UGACAUCUGGCUAAAAAGAAA UUUCUUUUUAGCCAGAUGUCA [2244-2264](21/21) 150 CUAGUUUCUGUGUAAGUGUAA UUACACUUACACAGAAACUAG [2156-2176](21/21) 151 GGCUAGUUUCUGUGUAAGUGU ACACUUACACAGAAACUAGCC [2154-2174](21/21) 152 UGGGCUAGUUUCUGUGUAAGU ACUUACACAGAAACUAGCCCA [2152-2172](21/21) 153 AAACAAUUUCUUAGGACACCA UGGUGUCCUAAGAAAUUGUUU [2129-2149](21/21) 154 GAGUGUCAGUAUGUUGAAUCA UGAUUCAACAUACUGACACUC [2089-2109](21/21) 155 UACUAAAAGCUCCUACUGUGA UCACAGUAGGAGCUUUUAGUA [1979-1999](21/21) 156 UGCUACGUGAUGAAGAUGGAA UUCCAUCUUCAUCACGUAGCA [1813-1833](21/21) 157 GCACCUUAUAUCUCGAAGUUU AAACUUCGAGAUAUAAGGUGC [1786-1806](21/21) 158 CAGCACCUUAUAUCUCGAAGU ACUUCGAGAUAUAAGGUGCUG [1784-1804](21/21) 159 CACCUACUGAAAAAACAACUC GAGUUGUUUUUUCAGUAGGUG [1764-1784](21/21) 160 AACUAGAGCAAGAUUUAGAUC GAUCUAAAUCUUGCUCUAGUU [1684-1704](21/21) 161 UAGUAGAACUAGAGCAAGAUU AAUCUUGCUCUAGUUCUACUA [1678-1698](21/21) 162 GGGAUAUACGUAGGAGGGGUA UACCCCUCCUACGUAUAUCCC [1609-1629](21/21) 163 UGAGAACACACCAGAGAAAGA UCUUUCUCUGGUGUGUUCUCA [1379-1399](21/21) 164 AAAGAGUAUGAGCUGGAAAAA UUUUUCCAGCUCAUACUCUUU [243-263](21/21) 165 CGGAAAGAGUAUGAGCUGGAA UUCCAGCUCAUACUCUUUCCG [240-260](21/21) 166 UGCCCCUGGAAGUGUCAAACA UGUUUGACACUUCCAGGGGCA [1262-1282](21/21) 167 AUAGUGCCCCUGGAAGUGUCA UGACACUUCCAGGGGCACUAU [1258-1278](21/21) 168 AGCGACGGAAAGAGUAUGAGC GCUCAUACUCUUUCCGUCGCU [235-255](21/21) 169 CAGCGACGGAAAGAGUAUGAG CUCAUACUCUUUCCGUCGCUG [234-254](21/21) 170 GGGCCCAUUGAUGUUUCUGAU AUCAGAAACAUCAAUGGGCCC [1056-1076](21/21) 171 CUGUAGUCCACAUUUUCUUAA UUAAGAAAAUGUGGACUACAG [836-856](21/21) 172 CUCAAUGGAAAAAGAAGUAGG CCUACUUCUUUUUCCAUUGAG [812-832](21/21) 173 GAUCUUGGAGUAAGUCGAGAA UUCUCGACUUACUCCAAGAUC [198-218](21/21) 174 CUUUGGAGGCAAGAUAUAGAU AUCUAUAUCUUGCCUCCAAAG [180-200](21/21) 175 GACAUUGAGCAAGUUUGGGAG CUCCCAAACUUGCUCAAUGUC [660-680](21/21) 176 GGACAUUGAGCAAGUUUGGGA UCCCAAACUUGCUCAAUGUCC [659-679](21/21) 177 GUCACCUGAAACUUCUGUUGC GCAACAGAAGUUUCAGGUGAC [602-622](21/21) 178 GCCCAGUCUUCAUUGCUACUA UAGUAGCAAUGAAGACUGGGC [571-591](21/21) 179 UCGAGAGCCCAGUCUUCAUUG CAAUGAAGACUGGGCUCUCGA [565-585](21/21) 180 UCGGCUACGUUUCAGUCACUU AAGUGACUGAAACGUAGCCGA [522-542](21/21) 181 GAGGUUUCUUCGGCUACGUUU AAACGUAGCCGAAGAAACCUC [513-533](21/21) 182 UUGUACUUUGAUGACUGCAUG CAUGCAGUCAUCAAAGUACAA [453-473](21/21) 183 AUGCUUUGUACUUUGAUGACU AGUCAUCAAAGUACAAAGCAU [448-468](21/21) 184 GAUGCUUUGUACUUUGAUGAC GUCAUCAAAGUACAAAGCAUC [447-467](21/21) 185 CUAGAUGAAGAGACAGGUGAA UUCACCUGUCUCUUCAUCUAG [339-359](21/21) 186 GUAUGGACAAAAAAUGGCAUU AAUGCCAUUUUUUGUCCAUAC [2306-2326](21/21) 187 UGUAUGGACAAAAAAUGGCAU AUGCCAUUUUUUGUCCAUACA [2305-2325](21/21) 188 ACAAUUUCUUAGGACACCAUU AAUGGUGUCCUAAGAAAUUGU [2131-2151](21/21) 189 UGAUGUGAAAUGCUCAUACUU AAGUAUGAGCAUUUCACAUCA [1997-2017](21/21) 190 UCCUACUGUGAUGUGAAAUGC GCAUUUCACAUCACAGUAGGA [1989-2009](21/21) 191 UCCUAGUGAAUACUCCCUGCA UGCAGGGAGUAUUCACUAGGA [1844-1864](21/21) 192 ACACGGUCCACAGCUCAUCAU AUGAUGAGCUGUGGACCGUGU [95-115](21/21) 193 GAAGUGUCAAACAGAAUGGUC GACCAUUCUGUUUGACACUUC [1270-1290](21/21) 194 UGGAAGUGUCAAACAGAAUGG CCAUUCUGUUUGACACUUCCA [1268-1288](21/21) 195 GUGAUUCUGAAGUGGAAGAGC GCUCUUCCACUUCAGAAUCAC [1234-1254](21/21) 196 GAAAGCACAGCAGAAUUCAAU AUUGAAUUCUGCUGUGCUUUC [1113-1133](21/21) 197 CACAGUCAACACAGAUUUUGG CCAAAAUCUGUGUUGACUGUG [938-958](21/21) 198 CAGAAGCCAAACUGACAGAAG CUUCUGUCAGUUUGGCUUCUG [760-780](21/21) 199 ACCUGAAACUUCUGUUGCUCA UGAGCAACAGAAGUUUCAGGU [605-625](21/21) 200 UUGACAUACUUUGGAGGCAAG CUUGCCUCCAAAGUAUGUCAA [172-192](21/21) 201 UUGAUUGACAUACUUUGGAGG CCUCCAAAGUAUGUCAAUCAA [168-188](21/21) 202 GUCACUUGUUCCUGAUAUUCC GGAAUAUCAGGAACAAGUGAC [536-556](21/21) 203 GCUUUGUACUUUGAUGACUGC GCAGUCAUCAAAGUACAAAGC [450-470](21/21) 204 CAGGACAUGGAUUUGAUUGAC GUCAAUCAAAUCCAUGUCCUG [156-176](21/21) 205 CAGUUACAACUAGAUGAAGAG CUCUUCAUCUAGUUGUAACUG [330-350](21/21) 206 GAUAUGACAUCUGGCUAAAAA UUUUUAGCCAGAUGUCAUAUC [2240-2260](21/21) 207 CAGUAUGUUGAAUCAGUAGUU AACUACUGAUUCAACAUACUG [2095-2115](21/21) 208 AGCAUUGGAGUGUCAGUAUGU ACAUACUGACACUCCAAUGCU [2082-2102](21/21) 209 AAAGCAUUGGAGUGUCAGUAU AUACUGACACUCCAAUGCUUU [2080-2100](21/21) 210 CCUACUGUGAUGUGAAAUGCU AGCAUUUCACAUCACAGUAGG [1990-2010](21/21) 211 UGACCUUUUCUGAGCUAGUUU AAACUAGCUCAGAAAAGGUCA [1945-1965](21/21) 212 GAUUUGACCUUUUCUGAGCUA UAGCUCAGAAAAGGUCAAAUC [1941-1961](21/21) 213 CAGCAUGCUACGUGAUGAAGA UCUUCAUCACGUAGCAUGCUG [1808-1828](21/21) 214 CACCUUAUAUCUCGAAGUUUU AAAACUUCGAGAUAUAAGGUG [1787-1807](21/21) 215 UCAGCACCUUAUAUCUCGAAG CUUCGAGAUAUAAGGUGCUGA [1783-1803](21/21) 216 ACUCAGCACCUUAUAUCUCGA UCGAGAUAUAAGGUGCUGAGU [1781-1801](21/21) 217 AUAGUAGAACUAGAGCAAGAU AUCUUGCUCUAGUUCUACUAU [1677-1697](21/21) 218 GGAUAUACGUAGGAGGGGUAA UUACCCCUCCUACGUAUAUCC [1610-1630](21/21) 219 AAAGCUCUCCAUAUCCCAUUC GAAUGGGAUAUGGAGAGCUUU [1497-1517](21/21) 220 GCAAAAGCUCUCCAUAUCCCA UGGGAUAUGGAGAGCUUUUGC [1494-1514](21/21) 221 AGAGAUGAACUUAGGGCAAAA UUUUGCCCUAAGUUCAUCUCU [1479-1499](21/21) 222 CAAGAGAUGAACUUAGGGCAA UUGCCCUAAGUUCAUCUCUUG [1477-1497](21/21) 223 UCACAAGAGAUGAACUUAGGG CCCUAAGUUCAUCUCUUGUGA [1474-1494](21/21) 224 GGAAGUGUCAAACAGAAUGGU ACCAUUCUGUUUGACACUUCC [1269-1289](21/21) 225 CCUGGAAGUGUCAAACAGAAU AUUCUGUUUGACACUUCCAGG [1266-1286](21/21) 226 GAUUCUGAAGUGGAAGAGCUA UAGCUCUUCCACUUCAGAAUC [1236-1256](21/21) 227 GCGACGGAAAGAGUAUGAGCU AGCUCAUACUCUUUCCGUCGC [236-256](21/21) 228 UUCUGAUCUAUCACUUUGCAA UUGCAAAGUGAUAGAUCAGAA [1070-1090](21/21) 229 AUCUUGGAGUAAGUCGAGAAG CUUCUCGACUUACUCCAAGAU [199-219](21/21) 230 CAACAGGACAUUGAGCAAGUU AACUUGCUCAAUGUCCUGUUG [654-674](21/21) 231 UCACCUGAAACUUCUGUUGCU AGCAACAGAAGUUUCAGGUGA [603-623](21/21) 232 AUCGAGAGCCCAGUCUUCAUU AAUGAAGACUGGGCUCUCGAU [564-584](21/21) 233 UACGUUUCAGUCACUUGUUCC GGAACAAGUGACUGAAACGUA [527-547](21/21) 234 GCUACGUUUCAGUCACUUGUU AACAAGUGACUGAAACGUAGC [525-545](21/21) 235 GGCUACGUUUCAGUCACUUGU ACAAGUGACUGAAACGUAGCC [524-544](21/21) 236 CGGCUACGUUUCAGUCACUUG CAAGUGACUGAAACGUAGCCG [523-543](21/21) 237 UUUCUUCGGCUACGUUUCAGU ACUGAAACGUAGCCGAAGAAA [517-537](21/21) 238 GACAUGGAUUUGAUUGACAUA UAUGUCAAUCAAAUCCAUGUC [159-179](21/21) 239 AUAUGACAUCUGGCUAAAAAG CUUUUUAGCCAGAUGUCAUAU [2241-2261](21/21) 240 UUGGGCUAGUUUCUGUGUAAG CUUACACAGAAACUAGCCCAA [2151-2171](21/21) 241 GUCAGUAUGUUGAAUCAGUAG CUACUGAUUCAACAUACUGAC [2093-2113](21/21) 242 UGUCAGUAUGUUGAAUCAGUA UACUGAUUCAACAUACUGACA [2092-2112](21/21) 243 GUGUCAGUAUGUUGAAUCAGU ACUGAUUCAACAUACUGACAC [2091-2111](21/21) 244 UUGGAGUGUCAGUAUGUUGAA UUCAACAUACUGACACUCCAA [2086-2106](21/21) 245 UACUGUGAUGUGAAAUGCUCA UGAGCAUUUCACAUCACAGUA [1992-2012](21/21) 246 ACCUUUUCUGAGCUAGUUUUU AAAAACUAGCUCAGAAAAGGU [1947-1967](21/21) 247 UUUGACCUUUUCUGAGCUAGU ACUAGCUCAGAAAAGGUCAAA [1943-1963](21/21) 248 UGCAGCAAACAAGAGAUGGCA UGCCAUCUCUUGUUUGCUGCA [1861-1881](21/21) 249 CUCCUAGUGAAUACUCCCUGC GCAGGGAGUAUUCACUAGGAG [1843-1863](21/21) 250 GCAUGCUACGUGAUGAAGAUG CAUCUUCAUCACGUAGCAUGC [1810-1830](21/21) 251 ACCUUAUAUCUCGAAGUUUUC GAAAACUUCGAGAUAUAAGGU [1788-1808](21/21) 252 UUAAUUCGGGAUAUACGUAGG CCUACGUAUAUCCCGAAUUAA [1602-1622](21/21) 253 CAACGAAAUGAUGUCCAAAGA UCUUUGGACAUCAUUUCGUUG [1556-1576](21/21) 254 AACCUCCCUGUUGUUGACUUC GAAGUCAACAACAGGGAGGUU [1536-1556](21/21) 255 GAGAUGAACUUAGGGCAAAAG CUUUUGCCCUAAGUUCAUCUC [1480-1500](21/21) 256 ACAAGAGAUGAACUUAGGGCA UGCCCUAAGUUCAUCUCUUGU [1476-1496](21/21) 257 AAAGCACAGCAGAAUUCAAUG CAUUGAAUUCUGCUGUGCUUU [1114-1134](21/21) 258 GAUGUUUCUGAUCUAUCACUU AAGUGAUAGAUCAGAAACAUC [1065-1085](21/21) 259 ACAGUCAACACAGAUUUUGGU ACCAAAAUCUGUGUUGACUGU [939-959](21/21) 260 UGAGGAUUCCUUCAGCAGCAU AUGCUGCUGAAGGAAUCCUCA [863-883](21/21) 261 UGACAUACUUUGGAGGCAAGA UCUUGCCUCCAAAGUAUGUCA [173-193](21/21) 262 CAUGGAUUUGAUUGACAUACU AGUAUGUCAAUCAAAUCCAUG [161-181](21/21) 263 GCAGGACAUGGAUUUGAUUGA UCAAUCAAAUCCAUGUCCUGC [155-175](21/21) 264 AUGAAGAGACAGGUGAAUUUC GAAAUUCACCUGUCUCUUCAU [343-363](21/21) 265 AGAUGAAGAGACAGGUGAAUU AAUUCACCUGUCUCUUCAUCU [341-361](21/21) 266 UUGCAAAACUAACCACUAUGU ACAUAGUGGUUAGUUUUGCAA [2268-2288](21/21) 267 AUGACAUCUGGCUAAAAAGAA UUCUUUUUAGCCAGAUGUCAU [2243-2263](21/21) 268 GGGCUAGUUUCUGUGUAAGUG CACUUACACAGAAACUAGCCC [2153-2173](21/21) 269 UUUGGGCUAGUUUCUGUGUAA UUACACAGAAACUAGCCCAAA [2150-2170](21/21) 270 GGAGUGUCAGUAUGUUGAAUC GAUUCAACAUACUGACACUCC [2088-2108](21/21) 271 UGUGAUGUGAAAUGCUCAUAC GUAUGAGCAUUUCACAUCACA [1995-2015](21/21) 272 CUCAGCACCUUAUAUCUCGAA UUCGAGAUAUAAGGUGCUGAG [1782-1802](21/21) 273 UAAUUCGGGAUAUACGUAGGA UCCUACGUAUAUCCCGAAUUA [1603-1623](21/21) 274 AAAGACAAGAACAACUCCAAA UUUGGAGUUGUUCUUGUCUUU [283-303](21/21) 275 GAAAGACAAGAACAACUCCAA UUGGAGUUGUUCUUGUCUUUC [282-302](21/21) 276 CGAAAUGAUGUCCAAAGAGCA UGCUCUUUGGACAUCAUUUCG [1559-1579](21/21) 277 AACGAAAUGAUGUCCAAAGAG CUCUUUGGACAUCAUUUCGUU [1557-1577](21/21) 278 ACCUCCCUGUUGUUGACUUCA UGAAGUCAACAACAGGGAGGU [1537-1557](21/21) 279 AUCAUUAACCUCCCUGUUGUU AACAACAGGGAGGUUAAUGAU [1530-1550](21/21) 280 UUUCUGAUCUAUCACUUUGCA UGCAAAGUGAUAGAUCAGAAA [1069-1089](21/21) 281 CACUUGUUCCUGAUAUUCCCG CGGGAAUAUCAGGAACAAGUG [538-558](21/21) 282 GAUUUGAUUGACAUACUUUGG CCAAAGUAUGUCAAUCAAAUC [165-185](21/21) 283 ACGUUUCAGUCACUUGUUCCU AGGAACAAGUGACUGAAACGU [528-548](21/21) 284 ACAGGUGAAUUUCUCCCAAUU AAUUGGGAGAAAUUCACCUGU [351-371](21/21) 285 GAGCUGGUACUAAUAAAGGAU AUCCUUUAUUAGUACCAGCUC [2372-2392](21/21) 286 CUGUGAUGUGAAAUGCUCAUA UAUGAGCAUUUCACAUCACAG [1994-2014](21/21) 287 GAAAAACUAGAUUUAGGAGGA UCCUCCUAAAUCUAGUUUUUC [1922-1942](21/21) 288 GCUCAAAGAAAAAGGAGAAAA UUUUCUCCUUUUUCUUUGAGC [1730-1750](21/21) 289 AGACAAGAACAACUCCAAAAG CUUUUGGAGUUGUUCUUGUCU [285-305](21/21) 290 UUCGGGAUAUACGUAGGAGGG CCCUCCUACGUAUAUCCCGAA [1606-1626](21/21) 291 AUGUCCAAAGAGCAGUUCAAU AUUGAACUGCUCUUUGGACAU [1566-1586](21/21) 292 UGAUGUCCAAAGAGCAGUUCA UGAACUGCUCUUUGGACAUCA [1564-1584](21/21) 293 GAGCUGGAAAAACAGAAAAAA UUUUUUCUGUUUUUCCAGCUC [252-272](21/21) 294 CCGGCAUUUCACUAAACACAA UUGUGUUUAGUGAAAUGCCGG [1144-1164](21/21) 295 UUGAUGUUUCUGAUCUAUCAC GUGAUAGAUCAGAAACAUCAA [1063-1083](21/21) 296 CCAUUGAUGUUUCUGAUCUAU AUAGAUCAGAAACAUCAAUGG [1060-1080](21/21) 297 CCCAUUGAUGUUUCUGAUCUA UAGAUCAGAAACAUCAAUGGG [1059-1079](21/21) 298 GGCCCAUUGAUGUUUCUGAUC GAUCAGAAACAUCAAUGGGCC [1057-1077](21/21) 299 AAGAAGUAGGUAACUGUAGUC GACUACAGUUACCUACUUCUU [823-843](21/21) 300 GACCUUUUCUGAGCUAGUUUU AAAACUAGCUCAGAAAAGGUC [1946-1966](21/21) 301 AUCUCGAAGUUUUCAGCAUGC GCAUGCUGAAAACUUCGAGAU [1795-1815](21/21) 302 AAGACAAGAACAACUCCAAAA UUUUGGAGUUGUUCUUGUCUU [284-304](21/21) 303 GAAAACCCCAUUCACAAAAGA UCUUUUGUGAAUGGGGUUUUC [1424-1444](21/21) 304 GAUAUGGUACAACCCUUGUCA UGACAAGGGUUGUACCAUAUC [1317-1337](21/21) 305 GAAGUGGAAGAGCUAGAUAGU ACUAUCUAGCUCUUCCACUUC [1242-1262](21/21) 306 GUUUCUGAUCUAUCACUUUGC GCAAAGUGAUAGAUCAGAAAC [1068-1088](21/21) 307 GCCCAUUGAUGUUUCUGAUCU AGAUCAGAAACAUCAAUGGGC [1058-1078](21/21) 308 UUGCCCACAUUCCCAAAUCAG CUGAUUUGGGAAUGUGGGCAA [427-447](21/21) 309 GUUGCCCACAUUCCCAAAUCA UGAUUUGGGAAUGUGGGCAAC [426-446](21/21) 310 GCAGCAAACAAGAGAUGGCAA UUGCCAUCUCUUGUUUGCUGC [1862-1882](21/21) 311 UGCUCAAAGAAAAAGGAGAAA UUUCUCCUUUUUCUUUGAGCA [1729-1749](21/21) 312 GAAGCUCAACUUGCAUUAAUU AAUUAAUGCAAGUUGAGCUUC [1587-1607](21/21) 313 CAAAGAGCAGUUCAAUGAAGC GCUUCAUUGAACUGCUCUUUG [1571-1591](21/21) 314 GGAAAACCCCAUUCACAAAAG CUUUUGUGAAUGGGGUUUUCC [1423-1443](21/21) 315 UGAGCUGGAAAAACAGAAAAA UUUUUCUGUUUUUCCAGCUCA [251-271](21/21) 316 GCCCAGUAUCAGCAACAGCAU AUGCUGUUGCUGAUACUGGGC [989-1009](21/21) 317 UAGGUAACUGUAGUCCACAUU AAUGUGGACUACAGUUACCUA [829-849](21/21) 318 CAUUCCUGAGUUACAGUGUCU AGACACUGUAACUCAGGAAUG [692-712](21/21) 319 CCGUUUGUAGAUGACAAUGAG CUCAUUGUCAUCUACAAACGG [495-515](21/21) 320 GCUGGUACUAAUAAAGGAUUA UAAUCCUUUAUUAGUACCAGC [2374-2394](21/21) 321 AGGACACCAUUUGGGCUAGUU AACUAGCCCAAAUGGUGUCCU [2141-2161](21/21) 322 GCUCUCCAUAUCCCAUUCCCU AGGGAAUGGGAUAUGGAGAGC [1500-1520](21/21) 323 UGGGCCCAUUGAUGUUUCUGA UCAGAAACAUCAAUGGGCCCA [1055-1075](21/21) 324 ACUUUAAGCCAUUCACUCUCU AGAGAGUGAAUGGCUUAAAGU [1023-1043](21/21) 325 CACCUGCUACUUUAAGCCAUU AAUGGCUUAAAGUAGCAGGUG [1015-1035](21/21) 326 CUCACCUGCUACUUUAAGCCA UGGCUUAAAGUAGCAGGUGAG [1013-1033](21/21) 327 UGAAAAUGACAAGCUGGUUGA UCAACCAGCUUGUCAUUUUCA [719-739](21/21) 328 AGCUGGUACUAAUAAAGGAUU AAUCCUUUAUUAGUACCAGCU [2373-2393](21/21) 329 AUGCAAAAUCAUAGCCAAAAC GUUUUGGCUAUGAUUUUGCAU [2031-2051](21/21) 330 CAAACAAGAGAUGGCAAUGUU AACAUUGCCAUCUCUUGUUUG [1866-1886](21/21) 331 GAAAAUGACAAAAGCCUUCAC GUGAAGGCUUUUGUCAUUUUC [1746-1766](21/21) 332 AAAACCCCAUUCACAAAAGAC GUCUUUUGUGAAUGGGGUUUU [1425-1445](21/21) 333 AUGAGCUGGAAAAACAGAAAA UUUUCUGUUUUUCCAGCUCAU [250-270](21/21) 334 CUGAAGUGGAAGAGCUAGAUA UAUCUAGCUCUUCCACUUCAG [1240-1260](21/21) 335 ACUCCGGCAUUUCACUAAACA UGUUUAGUGAAAUGCCGGAGU [1141-1161](21/21) 336 CCAUUCACUCUCUGAACUUCU AGAAGUUCAGAGAGUGAAUGG [1031-1051](21/21) 337 AGCCAUUCACUCUCUGAACUU AAGUUCAGAGAGUGAAUGGCU [1029-1049](21/21) 338 CUGCUACUUUAAGCCAUUCAC GUGAAUGGCUUAAAGUAGCAG [1018-1038](21/21) 339 UCACCUGCUACUUUAAGCCAU AUGGCUUAAAGUAGCAGGUGA [1014-1034](21/21) 340 UGCCCUCACCUGCUACUUUAA UUAAAGUAGCAGGUGAGGGCA [1009-1029](21/21) 341 AUGCCCUCACCUGCUACUUUA UAAAGUAGCAGGUGAGGGCAU [1008-1028](21/21) 342 AUGGAAAAAGAAGUAGGUAAC GUUACCUACUUCUUUUUCCAU [816-836](21/21) 343 GAAGCCAAACUGACAGAAGUU AACUUCUGUCAGUUUGGCUUC [762-782](21/21) 344 UUAAAAAGCAUUGGAGUGUCA UGACACUCCAAUGCUUUUUAA [2076-2096](21/21) 345 GGCAAUGUUUUCCUUGUUCCC GGGAACAAGGAAAACAUUGCC [1878-1898](21/21) 346 ACAACUCAGCACCUUAUAUCU AGAUAUAAGGUGCUGAGUUGU [1778-1798](21/21) 347 GGCUGCUCAGAAUUGCAGAAA UUUCUGCAAUUCUGAGCAGCC [1640-1660](21/21) 348 AGCUCUCCAUAUCCCAUUCCC GGGAAUGGGAUAUGGAGAGCU [1499-1519](21/21) 349 AAGCUCUCCAUAUCCCAUUCC GGAAUGGGAUAUGGAGAGCUU [1498-1518](21/21) 350 AAGACAAACAUUCAAGCCGCU AGCGGCUUGAAUGUUUGUCUU [1441-1461](21/21) 351 AAACCCCAUUCACAAAAGACA UGUCUUUUGUGAAUGGGGUUU [1426-1446](21/21) 352 CGGCAUUUCACUAAACACAAG CUUGUGUUUAGUGAAAUGCCG [1145-1165](21/21) 353 CCCGUUUGUAGAUGACAAUGA UCAUUGUCAUCUACAAACGGG [494-514](21/21) 354 UCCCGUUUGUAGAUGACAAUG CAUUGUCAUCUACAAACGGGA [493-513](21/21) 355 AUCUGCCAACUACUCCCAGGU ACCUGGGAGUAGUUGGCAGAU [407-427](21/21) 356 GCCAGAUGUUAAGAAAAACUA UAGUUUUUCUUAACAUCUGGC [1910-1930](21/21) 357 AAAUGACAAAAGCCUUCACCU AGGUGAAGGCUUUUGUCAUUU [1748-1768](21/21) 358 CUGCUCAGAAUUCCAGAAAAA UUUUUCUGCAAUUCUGAGCAG [1642-1662](21/21) 359 AAGCUCAACUUGCAUUAAUUC GAAUUAAUGCAAGUUGAGCUU [1588-1608](21/21) 360 GUCAUCGGAAAACCCCAUUCA UGAAUGGGGUUUUCCGAUGAC [1417-1437](21/21) 361 CCCCAACCAGUUGACAGUGAA UUCACUGUCAACUGGUUGGGG [899-919](21/21) 362 GAAAAAGAAGUAGGUAACUGU ACAGUUACCUACUUCUUUUUC [819-839](21/21) 363 UGGAAAAAGAAGUAGGUAACU AGUUACCUACUUCUUUUUCCA [817-837](21/21) 364 CAAGUCCAGAAGCCAAACUGA UCAGUUUGGCUUCUGGACUUG [754-774](21/21) 365 UGGGAGGAGCUAUUAUCCAUU AAUGGAUAAUAGCUCCUCCCA [675-695](21/21) 366 UGCAAAACUAACCACUAUGUA UACAUAGUGGUUAGUUUUGCA [2269-2289](21/21) 367 AUGGAAAACCUUAUUCUCCUA UAGGAGAAUAAGGUUUUCCAU [1828-1848](21/21) 368 AGCCUUCACCUACUGAAAAAA UUUUUUCAGUAGGUGAAGGCU [1758-1778](21/21) 369 AAGGAGAAAAUGACAAAAGCC GGCUUUUGUCAUUUUCUCCUU [1741-1761](21/21) 370 AAAGACAAACAUUCAAGCCGC GCGGCUUGAAUGUUUGUCUUU [1440-1460](21/21) 371 UAAGUCCUGGUCAUCGGAAAA UUUUCCGAUGACCAGGACUUA [1408-1428](21/21) 372 UAUGAGCUGGAAAAACAGAAA UUUCUGUUUUUCCAGCUCAUA [249-269](21/21) 373 ACAACCCUUGUCACCAUCUCA UGAGAUGGUGACAAGGGUUGU [1325-1345](21/21) 374 GUACAACCCUUGUCACCAUCU AGAUGGUGACAAGGGUUGUAC [1323-1343](21/21) 375 GACACACUACUUGGCCUCAGU ACUGAGGCCAAGUAGUGUGUC [1215-1235](21/21) 376 CUGAUCUAUCACUUUGCAAAG CUUUGCAAAGUGAUAGAUCAG [1072-1092](21/21) 377 CCUGCUACUUUAAGCCAUUCA UGAAUGGCUUAAAGUAGCAGG [1017-1037](21/21) 378 CGAGAAGUAUUUGACUUCAGU ACUGAAGUCAAAUACUUCUCG [213-233](21/21) 379 UUCCCGUUUGUAGAUGACAAU AUUGUCAUCUACAAACGGGAA [492-512](21/21) 380 GCGCAGACAUUCCCGUUUGUA UACAAACGGGAAUGUCUGCGC [483-503](21/21) 381 UGGAUCUGCCAACUACUCCCA UGGGAGUAGUUGGCAGAUCCA [404-424](21/21) 382 GCUCAGUUACAACUAGAUGAA UUCAUCUAGUUGUAACUGAGC [327-347](21/21) 383 GCAAAAUCAUAGCCAAAACUA UAGUUUUGGCUAUGAUUUUGC [2033-2053](21/21) 384 CUGAGCUAGUUUUUUUGUACU AGUACAAAAAAACUAGCUCAG [1954-1974](21/21) 385 GGAGGAUUUGACCUUUUCUGA UCAGAAAAGGUCAAAUCCUCC [1937-1957](21/21) 386 UAGGAGGAUUUGACCUUUUCU AGAAAAGGUCAAAUCCUCCUA [1935-1955](21/21) 387 GCUGCUCAGAAUUGCAGAAAA UUUUCUGCAAUUCUGAGCAGC [1641-1661](21/21) 388 GUGGCUGCUCAGAAUUGCAGA UCUGCAAUUCUGAGCAGCCAC [1638-1658](21/21) 389 AAGUGGCUGCUCAGAAUUGCA UGCAAUUCUGAGCAGCCACUU [1636-1656](21/21) 390 UGGUCCUAAAACACCAGUACA UGUACUGGUGUUUUAGGACCA [1286-1306](21/21) 391 GAAUGGUCCUAAAACACCAGU ACUGGUGUUUUAGGACCAUUC [1283-1303](21/21) 392 GGCCUCAGUGAUUCUGAAGUG CACUUCAGAAUCACUGAGGCC [1227-1247](21/21) 393 UCUUCCAGCUAUGGAGACACA UGUGUCUCCAUAGCUGGAAGA [1200-1220](21/21) 394 GGCAUUUCACUAAACACAAGU ACUUGUGUUUAGUGAAAUGCC [1146-1166](21/21) 395 GACUCCGGCAUUUCACUAAAC GUUUAGUGAAAUGCCGGAGUC [1140-1160](21/21) 396 UGCUACUUUAAGCCAUUCACU AGUGAAUGGCUUAAAGUAGCA [1019-1039](21/21) 397 GUAUUUGACUUCAGUCAGCGA UCGCUGACUGAAGUCAAAUAC [219-239](21/21) 398 UGAGCCCAGUAUCAGCAACAG CUGUUGCUGAUACUGGGCUCA [986-1006](21/21) 399 GAGAAGUAUUUGACUUCAGUC GACUGAAGUCAAAUACUUCUC [214-234](21/21) 400 UCGAGAAGUAUUUGACUUCAG CUGAAGUCAAAUACUUCUCGA [212-232](21/21) 401 CAGUGAACUCAUUAAAUUCAG CUGAAUUUAAUGAGUUCACUG [913-933](21/21) 402 AGAAGCCAAACUGACAGAAGU ACUUCUGUCAGUUUGGCUUCU [761-781](21/21) 403 GGGAGGAGCUAUUAUCCAUUC GAAUGGAUAAUAGCUCCUCCC [676-696](21/21) 404 UUGGGAGGAGCUAUUAUCCAU AUGGAUAAUAGCUCCUCCCAA [674-694](21/21) 405 GCCCCUGUUGAUUUAGACGGU ACCGUCUAAAUCAACAGGGGC [630-650](21/21) 406 GACAGGUGAAUUUCUCCCAAU AUUGGGAGAAAUUCACCUGUC [350-370](21/21) 407 UGCAAAAUCAUAGCCAAAACU AGUUUUGGCUAUGAUUUUGCA [2032-2052](21/21) 408 UCCCAAAAGUAAGAAGCCAGA UCUGGCUUCUUACUUUUGGGA [1895-1915](21/21) 409 UUGUUCCCAAAAGUAAGAAGC GCUUCUUACUUUUGGGAACAA [1891-1911](21/21) 410 ACAACUCCAAAAGGAGCAAGA UCUUGCUCCUUUUGGAGUUGU [293-313](21/21) 411 UCCCAUUCCCUGUAGAAAAAA UUUUUUCUACAGGGAAUGGGA [1510-1530](21/21) 412 UCAUCGGAAAACCCCAUUCAC GUGAAUGGGGUUUUCCGAUGA [1418-1438](21/21) 413 CAGAGAAAGAAUUGCCUGUAA UUACAGGCAAUUCUUUCUCUG [1390-1410](21/21) 414 CAGAGCACUCACGUGCAUGAU AUCAUGCACGUGAGUGCUCUG [1350-1370](21/21) 415 CACCAGUACAUUCUUCUGGGG CCCCAGAAGAAUGUACUGGUG [1297-1317](21/21) 416 UCCGGCAUUUCACUAAACACA UGUGUUUAGUGAAAUGCCGGA [1143-1163](21/21) 417 UGACUCCGGCAUUUCACUAAA UUUAGUGAAAUGCCGGAGUCA [1139-1159](21/21) 418 CCCUCACCUGCUACUUUAAGC GCUUAAAGUAGCAGGUGAGGG [1011-1031](21/21) 419 GUCCAGAAGCCAAACUGACAG CUGUCAGUUUGGCUUCUGGAC [757-777](21/21) 420 GCUGGUUGAGACUACCAUGGU ACCAUGGUAGUCUCAACCAGC [731-751](21/21) 421 UUCCUGAGUUACAGUGUCUUA UAAGACACUGUAACUCAGGAA [694-714](21/21) 422 CCCCUGUUGAUUUAGACCGUA UACCGUCUAAAUCAACAGGGG [631-651](21/21) 423 CCAGUGGAUCUGCCAACUACU AGUAGUUGGCAGAUCCACUGG [400-420](21/21) 424 AGACAGGUGAAUUUCUCCCAA UUGGGAGAAAUUCACCUGUCU [349-369](21/21) 425 UCGCUCAGUUACAACUAGAUG CAUCUAGUUGUAACUGAGCGA [325-345](21/21) 426 CCAGAUGUUAAGAAAAACUAG CUAGUUUUUCUUAACAUCUGG [1911-1931](21/21) 427 AGCCAGAUGUUAAGAAAAACU AGUUUUUCUUAACAUCUGGCU [1909-1929](21/21) 428 AUGGCAAUGUUUUCCUUGUUC GAACAAGGAAAACAUUGCCAU [1876-1896](21/21) 429 AGAGAUGGCAAUGUUUUCCUU AAGGAAAACAUUGCCAUCUCU [1872-1892](21/21) 430 GAACAACUCCAAAAGGAGCAA UUGCUCCUUUUGGAGUUGUUC [291-311](21/21) 431 GGGCAAAAGCUCUCCAUAUCC GGAUAUGGAGAGCUUUUGCCC [1492-1512](21/21) 432 AUCGGAAAACCCCAUUCACAA UUGUGAAUGGGGUUUUCCGAU [1420-1440](21/21) 433 ACGUGCAUGAUGCCCAAUGUG CACAUUGGGCAUCAUGCACGU [1360-1380](21/21) 434 GGUCCUAAAACACCAGUACAU AUGUACUGGUGUUUUAGGACC [1287-1307](21/21) 435 CAGUCAGCGACGGAAAGAGUA UACUCUUUCCGUCGCUGACUG [230-250](21/21) 436 CUCCGGCAUUUCACUAAACAC GUGUUUAGUGAAAUGCCGGAG [1142-1162](21/21) 437 GACUUCAGUCAGCGACGGAAA UUUCCGUCGCUGACUGAAGUC [225-245](21/21) 438 AAGCCAUUCACUCUCUGAACU AGUUCAGAGAGUGAAUGGCUU [1028-1048](21/21) 439 CUGAGCCCAGUAUCAGCAACA UGUUGCUGAUACUGGGCUCAG [985-1005](21/21) 440 GACAGUGAACUCAUUAAAUUC GAAUUUAAUGAGUUCACUGUC [911-931](21/21) 441 GAAGACCCCAACCAGUUGACA UGUCAACUGGUUGGGGUCUUC [894-914](21/21) 442 AGCCAAACUGACAGAAGUUGA UCAACUUCUGUCAGUUUGGCU [764-784](21/21) 443 AAGCUGGUUGAGACUACCAUG CAUGGUAGUCUCAACCAGCUU [729-749](21/21) 444 GAGGAGCUAUUAUCCAUUCCU AGGAAUGGAUAAUAGCUCCUC [678-698](21/21) 445 GGUCCACAGCUCAUCAUGAUG CAUCAUGAUGAGCUGUGGACC [99-119](21/21) 446 GCUACUAAUCAGGCUCAGUCA UGACUGAGCCUGAUUAGUAGC [585-605](21/21) 447 CCCAGUCUUCAUUGCUACUAA UUAGUAGCAAUGAAGACUGGG [572-592](21/21) 448 UGAUGACUGCAUGCAGCUUUU AAAAGCUGCAUGCAGUCAUCA [461-481](21/21) 449 GAGACAGGUGAAUUUCUCCCA UGGGAGAAAUUCACCUGUCUC [348-368](21/21) 450 UUUCGCUCAGUUACAACUAGA UCUAGUUGUAACUGAGCGAAA [323-343](21/21) 451 AUGAUAUGACAUCUGGCUAAA UUUAGCCAGAUGUCAUAUCAU [2238-2258](21/21) 452 GGACACCAUUUGGGCUAGUUU AAACUAGCCCAAAUGGUGUCC [2142-2162](21/21) 453 UCAUAGCCAAAACUAGUAUAG CUAUACUAGUUUUGGCUAUGA [2039-2059](21/21) 454 UCUGAGCUAGUUUUUUUGUAC GUACAAAAAAACUAGCUCAGA [1953-1973](21/21) 455 GAGAUGGCAAUGUUUUCCUUG CAAGGAAAACAUUGCCAUCUC [1873-1893](21/21) 456 GUGAAUACUCCCUGCAGCAAA UUUGCUGCAGGGAGUAUUCAC [1849-1869](21/21) 457 AACCUUAUUCUCCUAGUGAAU AUUCACUAGGAGAAUAAGGUU [1834-1854](21/21) 458 GGAAAACCUUAUUCUCCUAGU ACUAGGAGAAUAAGGUUUUCC [1830-1850](21/21) 459 GCCUUCACCUACUGAAAAAAC GUUUUUUCAGUAGGUGAAGGC [1759-1779](21/21) 460 UGCUCAGAAUUGCAGAAAAAG CUUUUUCUGCAAUUCUGAGCA [1643-1663](21/21) 461 GAGUAUGAGCUGGAAAAACAG CUGUUUUUCCAGCUCAUACUC [246-266](21/21) 462 AUGGUCCUAAAACACCAGUAC GUACUGGUGUUUUAGGACCAU [1285-1305](21/21) 463 ACACACUACUUGGCCUCAGUG CACUGAGGCCAAGUAGUGUGU [1216-1236](21/21) 464 GAGACACACUACUUGGCCUCA UGAGGCCAAGUAGUGUGUCUC [1213-1233](21/21) 465 AUGGAGACACACUACUUGGCC GGCCAAGUAGUGUGUCUCCAU [1210-1230](21/21) 466 AGCUAUGGAGACACACUACUU AAGUAGUGUGUCUCCAUAGCU [1206-1226](21/21) 467 GCAUUUCACUAAACACAAGUC GACUUGUGUUUAGUGAAAUGC [1147-1167](21/21) 468 UCAGUCAGCGACGGAAAGAGU ACUCUUUCCGUCGCUGACUGA [229-249](21/21) 469 AUUCACUCUCUGAACUUCUAA UUAGAAGUUCAGAGAGUGAAU [1033-1053](21/21) 470 AACACAGAUUUUGGUGAUGAA UUCAUCACCAAAAUCUGUGUU [945-965](21/21) 471 GUCAACACAGAUUUUGGUGAU AUCACCAAAAUCUGUGUUGAC [942-962](21/21) 472 GAACUCAUUAAAUUCAGAUGC GCAUCUGAAUUUAAUGAGUUC [917-937](21/21) 473 ACCAGUUGACAGUGAACUCAU AUGAGUUCACUGUCAACUGGU [904-924](21/21) 474 AGGUAACUGUAGUCCACAUUU AAAUGUGGACUACAGUUACCU [830-850](21/21) 475 UAUACCCUCAAUGGAAAAAGA UCUUUUUCCAUUGAGGGUAUA [806-826](21/21) 476 UCCAUUCCUGAGUUACAGUGU ACACUGUAACUCAGGAAUGGA [690-710](21/21) 477 GGAGCUAUUAUCCAUUCCUGA UCAGGAAUGGAUAAUAGCUCC [680-700](21/21) 478 ACGGUAUGCAACAGGACAUUG CAAUGUCCUGUUGCAUACCGU [646-666](21/21) 479 CCCUGUUGAUUUAGACGGUAU AUACCGUCUAAAUCAACAGGG [632-652](21/21) 480 GUAGCCCCUGUUGAUUUAGAC GUCUAAAUCAACAGGGGCUAC [627-647](21/21) 481 GAAACUUCUGUUGCUCAGGUA UACCUGAGCAACAGAAGUUUC [609-629](21/21) 482 GAGAGCCCAGUCUUCAUUGCU AGCAAUGAAGACUGGGCUCUC [567-587](21/21) 483 AAACCAGUGGAUCUGCCAACU AGUUGGCAGAUCCACUGGUUU [397-417](21/21) 484 UUAGGAGGAUUUGACCUUUUC GAAAAGGUCAAAUCCUCCUAA [1934-1954](21/21) 485 AAGCCAGAUGUUAAGAAAAAC GUUUUUCUUAACAUCUGGCUU [1908-1928](21/21) 486 UUCCCAAAAGUAAGAAGCCAG CUGGCUUCUUACUUUUGGGAA [1894-1914](21/21) 487 GGAGCAAGAGAAAGCCUUUUU AAAAAGGCUUUCUCUUGCUCC [305-325](21/21) 488 GAUGGCAAUGUUUUCCUUGUU AACAAGGAAAACAUUGCCAUC [1875-1895](21/21) 489 AAGGAGCAAGAGAAAGCCUUU AAAGGCUUUCUCUUGCUCCUU [303-323](21/21) 490 AAGCCUUCACCUACUGAAAAA UUUUUCAGUAGGUGAAGGCUU [1757-1777](21/21) 491 UGAAGCUCAACUUGCAUUAAU AUUAAUGCAAGUUGAGCUUCA [1586-1606](21/21) 492 AUGAAGCUCAACUUGCAUUAA UUAAUGCAAGUUGAGCUUCAU [1585-1605](21/21) 493 CCCAUUCCCUGUAGAAAAAAU AUUUUUUCUACAGGGAAUGGG [1511-1531](21/21) 494 UUGGAGGCUCAUCUCACAAGA UCUUGUGAGAUGAGCCUCCAA [1461-1481](21/21) 495 AGCUGGAAAAACAGAAAAAAC GUUUUUUCUGUUUUUCCAGCU [253-273](21/21) 496 GUGCAUGAUGCCCAAUGUGAG CUCACAUUGGGCAUCAUGCAC [1362-1382](21/21) 497 CACGUGCAUGAUGCCCAAUGU ACAUUGGGCAUCAUGCACGUG [1359-1379](21/21) 498 ACACCAGUACAUUCUUCUGGG CCCAGAAGAAUGUACUGGUGU [1296-1316](21/21) 499 GUCCUAAAACACCAGUACAUU AAUGUACUGGUGUUUUAGGAC [1288-1308](21/21) 500 UGGAGACACACUACUUGGCCU AGGCCAAGUAGUGUGUCUCCA [1211-1231](21/21)

Example 2 In Vitro Testing of siRNA Compounds for Nrf2 1. General

1.5-2×10⁵ tested cells (HeLa or 293 cells) were seeded per well in a 6-well plate (70-80% confluent).

24 h subsequently, cells were transfected with siRNA oligos using lipofectamine 2000 reagent (Invitrogene) at a final concentration of 500 μM, 5 nM, 20 nM or 40 W. The cells were incubated at 37° C. in a CO2 incubator for 72 h.

As positive control for cell transfection, PTEN-Cy3 labeled siRNA oligos were used.

As negative control for siRNA activity, GFP siRNA oligos were used.

72 h after transfection cells were harvested and RNA was extracted from cells.

Transfection efficiency was tested by fluorescent microscopy.

Results:

The percent of inhibition of gene expression using specific preferred siRNAs was determined using qPCR analysis of target gene in cells expressing the endogenous gene. The data in Table C demonstrate the percent of knockdown of the expression of the target gene in cells. In general, the siRNAs having specific sequences that were selected for in vitro testing were specific for both human and the rat/rabbit genes. Similar results of reduced expression of specific genes are obtained with other siRNAs, the sequences of which are listed in Tables A and B.

TABLE C Percent of knockdown of the expression of the target Nrf2 human gene in cells using siRNAs having preferred sequences (results of separate experiments are indicated). % knockdown of Active siRNA Sequence the human gene NFE2L2_10 Sense: 20 nM in HeLa cells: (Sense SEQ ID NO: 501) CCCUGUCGAAAAAAUCAUU 91%, 54%, 51% having one mismatch (in bold) Antisense: inhibition compared to the human AAUGAUUUUUUCGACAGGG 5 nM in HeLa cells: sequence 91% inhibition NFE2L2_4 Sense: 20 nM - 80%, 41% (Sense SEQ ID NO: 119) CCCAUUCACAAAAGACAAA inhibition Antisense: 5 nM - 73%, 36% UUUGUCUUUUGUGAAUGGG inhibition NFE2L2_5 Sense: 20 nM - 69%, 17% (Sense SEQ ID NO: 195): CAGCAGGACAUGGAUUUGA inhibition Antisense: 5 nM - 47%, 16% UCAAAUCCAUGUCCUGCUG inhibition NFE2L2_9 Sense: 20 nM - 44% (Sense SEQ ID NO: 502) UCCCUGUCGAAAAAAUCAU inhibition Antisense: 5 nM - 25% AUGAUUUUUUCGACAGGGA inhibition NFE2L2_11 Sense: 20 nM - 46% (Sense SEQ ID NO: 503) CCUGUCGAAAAAAUCAUUA inhibition Antisense: 5 nM - 23% UAAUGAUUUUUUCGACAGG inhibition NFE2L2_1 Sense: 20 nM - 55%, 73%, (Sense SEQ ID NO: 2) GGAGGGGUAAGAAUAAAGU 53% inhibition Antisense: ACUUUAUUCUUACCCCUCC NFE2L2_2 Sense: 20 nM - 92%, 49%, (Sense SEQ ID NO: 7) GCCCUCACCUGCUACUUUA 46% inhibition Antisense: UAAAGUAGCAGGUGAGGGC NFE2L2_3 Sense: 20 nM - 96%, 83%, (Sense SEQ ID NO: 10) UCCCGUUUGUAGAUGACAA 82% inhibition Antisense: UUGUCAUCUACAAACGGGA NFE2L2_12 Sense: 20 nM - 95%, 96%, (Sense SEQ ID NO: 26) GUAAGAAGCCAGAUGUUAA 76% inhibition Antisense: UUAACAUCUGGCUUCUUAC

Example 3 The Effect of Nrf2 siRNA Treatment on Tumor Growth In Vivo Methods:

Tumor Xenografts: A549 cells (5×10⁶) were injected into the hind leg of male athymic nude mice and the tumor was measured weekly. The tumor volumes were measured using the following formula: [length (mm)×width (mm)×width (mm)×0.52]. In the lung metastasis experiments, 2×106 A549-C8-luc cells were injected into SCID-Beige mice (Charles River, Mass.) intravenously.

For the in vivo experiments, all siRNA compounds were chemically synthesized being stabilized by alternating 2-O′-Me modifications. The sequence of siRNA targeting human Nrf2 used for in vivo experiments is 5′-UCCCGUUUGUAGAUGACAA-3′ (sense, SEQ ID NO:10) and 5′-UUGUCAUCUACAAACGGGA-3′ (antisense, SEQ ID NO:513). The sequence of control siRNA targeting GFP is 5′-GGCUACGUCCAGGAGCGCACC-3′ (sense, SEQ ID NO: 2007) and 5′-GGUGCGCUCCUGGACGUAGCC-3′ (antisense, SEQ ID NO: 2008).

For in vivo delivery of siRNA into subcutaneous tumors, siRNA duplexes diluted in PBS were injected into the hind leg tumors using insulin syringes at a concentration of 10 μg/mm³. Intraperitoneal injections of carboplatin were given at a dose of 40 mg/kg body weight. Both siRNA and carboplatin were administered twice weekly for 4 weeks.

For lung tumor delivery, female C57B6 mice were injected with Lewis Lung Carcinoma (LLC) cells (0.5×10⁶) intravenously, 24 days prior to the delivery experiment. Upon development of lung metastases, mice were administered with 100 μg/mouse of Cy3-labeled naked chemically stabilized reference siRNA via nebulizer inhalation on 3 consequent days. Mice were euthanized 24 hrs after the last inhalation. Upon termination, lungs were inflated with ice-cold 4% paraformaldehyde, followed by manual sectioning with razor blades. Clearly visible large surface tumors were sectioned separately. Resulting sections were analyzed by Bio-Rad Confocal microscope using a 20× Water objective and 2× zoom combined to give a total of 40× magnification. Control, non-siRNA-treated lungs were used to set up background fluorescence level.

For aerosol delivery of Nrf2 or GFP siRNA into lung tumors, 100 μg of siRNA duplex diluted in PBS was aerosolized using a nebulizer. Mice were given three dose of siRNA (100 μg/dose) every week, for 4 weeks, using a nebulizer. Intraperitoneal injections of carboplatin were given at a dose of 30 mg/kg body weight twice/week.

In Vivo Imaging: Animals inoculated with A549-C8-luc cells, which express a luciferase reporter gene, were anesthetized and injected intraperitoneally with 250 ul of luminescent substrate (15 mg/ml stock) D-Luciferin Firefly (Xenogen Cat# XR-1001). The animals were then imaged and analyzed by using the Xenogen IVIS Optical Imaging Device in the Johns Hopkins Oncology Center.

Statistical Analysis—Statistical comparisons were performed by Student's t-tests. A value of p<0.05 was considered statistically significant. Tumor weights and changes in tumor volume were summarized using descriptive statistics. Differences in tumor measures between treatment groups were examined using linear regression models with generalized estimating equations (GEE). The distributions of both tumor measurements were skewed, so log transformations were used.

Results:

A tumor xenograft experiment with A549 cells which were injected into the hind leg was conducted in order to test the anti-tumor activity of Nrf2 siRNA in vivo. Mice bearing subcutaneous tumors were treated with Nrf2 siRNA (comprising SEQ ID NO: 10) by direct injection into the tumor and by carboplatin twice a week for 4 weeks and tumor weight was measured at the termination of the experiment. Treatment with control non-targeting siRNA (GFP siRNA) did not inhibit tumor growth as compared to control mice treated with PBS alone. The tumor weights were significantly different between GFP siRNA and Nrf2 siRNA treated tumors (P=0.0002). Treatment with Nrf2 siRNA alone reduced mean tumor weight by 53% (±20% SD) compared to the control group. When Nrf2 siRNA was combined with carboplatin, there was an even greater reduction in mean tumor weight in all animals. To explore the effect of radiation exposure in combination with Nrf2 inhibition in vivo, the same A549 cell tumor xenograft model was used. In comparison with control siRNA+ionizing radiation (IR) treated tumors, combination of Nrf2 siRNA+IR (2 fractions of 3Gy irradiation) produced an additive effect on tumor growth inhibition (Tables D1 and D2). Delivery of naked Nrf2 siRNA duplex into tumor inhibited the expression of Nrf2 and its downstream target genes (HO-1 and GCLm), P<0.05 (FIG. 1).

TABLE D1 Mean (SD) of tumor weights and changes in tumor volume in tumor xenograft mice following treatment with Nrf2 siRNA (SEQ ID NO: 10), carboplatin, radiation and combination thereof (Experiment 1). Mean (SD) change in Mean (SD) tumor Treatment tumor volume (mm³) weight (mg) GFP siRNA 532.94 (260.94)  426 (187.83) GFP siRNA + 246.77 (189.39) 341.25 (275.27)   radiation GFP siRNA + 352.34 (170.96) 212 (66.48) carboplatin Nrf2 siRNA 249.17 (111.37) 238 (64.58) Nrf2 siRNA + 116.04 (147.9)  118 (70.85) radiation Nrf2 siRNA + 58.78 (80.06) 110 (57.62) carboplatin

TABLE D2 Mean (SD) of tumor weights and changes in tumor volume in tumor xenograft mice following treatment with Nrf2 siRNA (SEQ ID NO: 10), carboplatin, radiation and combination thereof (Experiment 2). Mean (SD) change in Mean (SD) tumor Treatment tumor volume (mm³) weight (mg) GFP siRNA   375 (233.7)  327.5 (201.56) GFP siRNA + 187.33 (76.66)    185 (67.16) carboplatin Nrf2 siRNA 138.67 (128.24) 143.33 (90.74)  Nrf2 siRNA +   45 (33.81) 58.33 (21.37) carboplatin

The delivery of naked siRNA duplexes into orthotopic lung tumors was examined. Mice were injected with Lewis lung carcinoma cells and 24 days later (when the mice developed larger tumors) mice were inhaled for three consecutive days with 100 μg/day/mouse of Cy3 labeled naked chemically stabilized reference siRNA using a nebulizer. Twenty four hours after last siRNA administration, mice were sacrificed; lungs harvested and sectioned. Resulting sections were analyzed by Bio-Rad Confocal microscope using a 20× Water objective and 2× zoom combined to give a total of 40× magnification. Control, non-siRNA-treated lungs were used to set up background fluorescence level. The fluorescence results revealed localization of Cy3 labeled siRNA in a large surface tumor, especially in intraparenchymal tumor. The large surface-protruding tumors showed Cy3 signal but the intensity was several folds lower than that observed in the small intra-parenchymal tumors.

In the next step, mice with orthotopic growing tumors expressing ARE-Luc Nrf2-dependent reporter were used. The mice with A549 lung tumors were treated with Nrf2 siRNA (SEQ ID NO: 10) intranasally using a nebulizer followed by carboplatin treatment. As demonstrated in FIG. 2, Nrf2 siRNA administered intranasally inhibited Nrf2 reporter activity in vivo, indicating specific inhibition of Nrf2 expression using Nrf2 siRNA following intranasal administration. Mice receiving Nrf2 siRNA together with carboplatin demonstrated higher growth inhibition as compared with control mice receiving GFP siRNA together with carboplatin (Table D3). The mean lung weight at termination and the luminescent flux intensity (evaluated by in vivo imaging) were lowest in mice treated with Nrf2 siRNA+carboplatin. Thus, combination of Nrf2 siRNA with carboplatin/radiation led to a reduction in tumor growth after 4 weeks of treatment compared with either agent alone. This study suggests that Nrf2 siRNA inhibitors may be considered as highly efficient promoters for the antineoplastic potential of platinum drugs such as carboplatin, causing additive/synergistic effects in lung cancer cells.

TABLE D3 Mean (SD) of lung tumor weights following intranasal treatment with Nrf2 siRNA (SEQ ID NO: 10) alone, and in combination with carboplatin. Treatment Mean (SD) tumor weight (mg) GFP siRNA 710 (167.93) GFP siRNA + 480 (95.13) carboplatin Nrf2 siRNA 802 (172.25) Nrf2 siRNA + 370 (62.05) carboplatin Vehicle siRNA 760 (98.99) Vehicle siRNA + 495 (49.5) carboplatin 

1. A compound having the structure: 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y) 5′ (sense strand)

wherein each N and N′ is a ribonucleotide which may independently be modified or unmodified in its sugar residue; wherein each of (N)_(x) and (N′)_(y) is an oligomer in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond; wherein each of x and y is an integer between 19 and 40; wherein each of Z and Z′ may be present or absent, but if present is 1-5 consecutive nucleotides covalently attached at the 3′ terminus of the strand in which it is present; and wherein the sequence of (N)_(x) is set forth as any one of SEQ ID NOS: 504-1006 or 1507-2006 and the sequence of (N)_(y) comprises a sequence complementary to (N)_(x) set forth in any one of SEQ ID NOS: 1-503 and 1007-1506, respectively,
 2. The compound of claim 1, wherein the covalent bond is a phosphodiester bond.
 3. The compound of claim 2, wherein x=y=19.
 4. The compound of claim 3, wherein Z and Z′ are both absent.
 5. The compound of claim 3, wherein one of Z or Z′ is present.
 6. The compound of claim 1, wherein all of the ribonucleotides in (N)_(x) and (N′)_(y) or both are unmodified in their sugar residues.
 7. The compound of claim 1, wherein at least one ribonucleotide in (N)_(x) and (N′)_(y) or both is modified in its sugar residue.
 8. The compound of claim 7, wherein the modification comprises a modification at the 2′ position of the sugar residue.
 9. The compound of claim 8, wherein the modification at the 2′ position of the sugar residue results in the presence of a moiety selected from the group comprising amino, fluoro, methoxy, alkoxy and alkyl groups.
 10. The compound of claim 24, wherein the alkoxy group is a methoxy (2′-O-methyl) group.
 11. The compound of claim 10, wherein alternating ribonucleotides in the antisense strand or in the sense strand or both strands are modified in their sugar residues.
 12. The compound of claim 11, wherein each ribonucleotide at the 5′ and 3′ termini of the antisense strand is modified in its sugar residues, and each ribonucleotide at the 5′ and 3′ termini of the sense strand is unmodified in its sugar residues.
 13. The compound of claim 12, wherein the antisense and the sense strands are non-phosphorylated at the 3′ and 5′ termini or wherein the antisense and the sense strands are phosphorylated at the 3′ termini.
 14. (canceled)
 15. A composition comprising a pharmaceutically acceptable carrier and a compound of claim
 1. 16. A method of treating a patient suffering from a disorder comprising administering to the patient the compound of claim 1 in a therapeutically effective dose thereby treating the patient.
 17. The method of claim 16, wherein the disorder is a cancerous disease selected from the group consisting of lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer.
 18. The method of claim 17, wherein the cancerous disease is lung cancer.
 19. The method of claim 18, wherein the composition comprises a naked siRNA compound.
 20. The method of claim 19, wherein the composition is administered via aerosol directly to the inner lung of the patient.
 21. The method of claim 20, further comprising administering to the patient a chemotherapy drug.
 22. (canceled)
 23. (canceled)
 24. The compound of claim 9, wherein the modification at the 2′ position of the sugar residue comprises the presence of an alkoxy group. 